Pinus halepensis seedlings during an induced moderate drought, two four-day-long heatwaves, and the combination of drought and heatwaves. We found clear stress-specific responses of BVOC emissions. Reductions in acetone emissions with declining soil water content and transpiration stood out as a clear drought indicator. All other measured BVOC emissions responded exponentially to rising temperatures during heat stress (maximum of 43 °C), but monoterpenes and methyl salicylate showed a reduced temperature sensitivity during the second heatwave. We found that these decreases in monoterpene emissions between heatwaves were not reflected by similar declines in their internal storage pools. Because stress intensity was extremely severe, most of the seedlings in the heat-drought treatment died at the end of the second heatwave (dark respiration ceased). Interestingly, BVOC emissions  differed between dying and surviving seedlings, already well before indications of a reduced vitality became visible in gas exchange dynamics. In summary, we could clearly show that the dynamics of BVOC emissions are sensitive to stress type, stress frequency, and stress severity. Moreover, we found indications that stress-induced seedling mortality was preceded by altered methanol, monoterpene, and acetaldehyde emission dynamics.Biogenic volatile organic compounds (BVOC) play important roles in plant stress responses and can serve as stress indicators. While the impacts of gradual environmental changes on BVOCs have been studied extensively, insights in emission responses to repeated stress and recovery are widely absent. Therefore, we studied the dynamics of shoot gas exchange and BVOC emissions in The online version contains supplementary material available at 10.1007/s00442-021-04905-y. Climate change is expected to cause not only higher temperatures and a higher variability of precipitation, but also to produce more frequent and more intense extreme events such as heatwaves and drought spells  and eventually limits photosynthesis (Anet)   of the surrounding air, which then leads to an increase in water loss by transpiration  is another response to cope with abiotic stress  seedlings were cultivated from seeds in a controlled greenhouse environment in Garmisch-Partenkirchen, Germany . Seed material originated from five different trees in a 55-year-old Aleppo pine plantation in Israel . The genetic heritage is not clear (Schiller and Atzmon n = 10 per treatment). Per treatment, four seedlings each were equipped with a gas exchange cuvette . Therefore, iteration of the seedlings’ placement during the experimental phase was no longer possible.Aleppo pine  Fig. S1). The heatwaves were designed to mimic naturally occurring few day-long heatwaves in the Yatir forest typically occurring during early summer  were distributed spatially within each greenhouse compartment and their shoots were placed permanently in transparent cuvettes  made from acrylic glass tubes . Dismountable acrylic glass caps on the downward-facing side allowed to install the cuvette at the seedlings stem. The inside of the cuvettes had been coated with chemical inert foil FEP , Small gaps between the stem and the cuvette were sealed with plastic putty  to minimize gas leakage. A fan  guaranteed well-mixing of the air inside the cuvettes. To assess environmental conditions, each cuvette was equipped with a calibrated photo diode , a calibrated thermocouple .A custom-made, open chamber system, which has been previously described  were measured continuously in an automated sequence as follows. After the distal cap of the cuvette had closed, a constant air stream  of clean air with on average 438 ± 3 µmol mol−1 [CO2] and 6.5 ± 0.1 mmol mol−1 [H2O] was supplied for 10 min. Zero air was generated by using an oil-free compressor  connected to an Ultra Zero Air generator . CO2 was supplied from a gas cylinder and water vapour was added to the air stream via a nebulizing evaporation pump . The air supply was channeled through the main tubing made of stainless-steel tubing  coated with SilcoNert  and gas flow to the cuvettes was controlled by two 2/2-way solenoid valves with a PTFE housing  and PTFE tubing . The detailed schematic of the measurement system can be found in Bamberger et al. . Net CO2 exchange , transpiration (E) and stomatal conductance (gs) were calculated as previously described in Birami et al. (−1) and quantified using a defined VOC mixture (14 components in nitrogen) of standard gas . PTR-MS calibration was performed at ambient humidity with a liquid calibration unit  on a weekly basis using a four-step calibration routine at mole fractions of 7.5, 5, 2.5 and 0 ppb.Differences in [COi et al. . BVOC fli et al. . Volatil6H10O on m z−1 99 was estimated to be on average 3.15 ± 0.13 ncps ppb−1, derived from the average sensitivity of xylene and toluene (the compound in the standard mixture being closest to m z−1 99 to consider for the transmission efficiency of the quadrupole mass filter) multiplied by 0.33, its known fractionation patterns (33% on m z−1 99) . The limit of detection for m z−1 99 is hence not given. The sensitivity for C8H8O3 m z−1153 (on average 4.96 ± 0.33 ncps ppb−1) was derived from a liquid calibration using a calibration mixture of methyl salicylate  in H2O  . Isoprene could not be distinguished from 2-methyl-3-butene-2-ol (MBO) with our method, hence we did not investigate this compound in detail and address it as “isoprene + MBO”. Ethanol could not be detected in satisfactory quality and was hence not further interpreted , given that the stability quality criteria were full-filled and backflow from the cuvettes was > 0.3 L min−1. Linear regression data were used to assess stability of the signal using an upper boundary for the regression slope ≤ 3 Eν in nmol m−2 s−1) were calculated from the concentration differences of air leaving the cuvette cout and the concentrations leaving an empty chamber c0  and extracted at 4 °C for 3 h in continuous shaking. Samples were incubated overnight at 5 °C, and the supernatant was transferred into a new 2 mL glass vial using a 1 mL gas-tight syringe (Hamilton). Finally, 1 µL per sample was analyzed by thermo-desorption (TD) gas chromatography mass spectrometry (GC–MS)  were immediately frozen in liquid nitrogen and ground in porcelain mortars to a fine powder and stored at  – 80 °C until further processing. MT and SQT were analyzed similarly as done before  were performed to assess if the variability explained by the model is higher than the variability explained by a randomly generated set of variables ) between normalized emission rates, treatment and experimental period. CCA allows to test for the significance of each of the ordination axis, as well as for the influence of factors and cofactors. Monte Carlo permutations (here  Oksanen . BVOC em Oksanen : (i) pre Oksanen .2 was calculated both for the fixed and the combined fixed and random effects and confidence intervals (CI) given  were used to test for dependencies of BVOC emissions, gas exchange on rSWC or temperature and treatment (fixed effects) Seedling was considered as a random effect. Temporal auto-correlation was accounted for by including a first-order Auto-Regressive model in the lme . The impact of dying and death on BVOC emissions was analyzed by grouping the heat and heat-drought seedlings into surviving (n = 4) and dying (n = 4). In case to overcome tree-specific differences in absolute emission rates, we normalized the data to a tree-specific median derived from before stress-conditions (110–114 DOY), which was for instance done when analyzing mortality responses. Treatment-averaged absolute emission rates per experimental period can be found in the Supplement (Table S4) and are given in Fig. During the course of the experiment, three of the continuously measured seedlings  in the heat-drought and one in the heat-treatment died due to overheating  treatment responded similar to heat stress  than by gs (pseudo-R2fixed = 0.1). Hence, changes in acetone emission were most likely caused by soil water availability and tree water flux. As we observed that acetone emissions from our potting-medium were negligible , acetone was most likely produced in the root tissues and transported to the shoots via the transpiration stream. No other BVOC showed a clear drought response -0.85), R2 = 0.94 of log-transformed function, Fig. S3b), while gs decreased to tightly regulate seedling water loss. In contrast, all observed BVOC emissions, except acetone  followed an exponential temperature response with slightly lower transpiration rates during the second heatwave.We investigated the impact of repeating heatwaves on the temperature response of BVOC emissions and gas exchange in the surviving Aleppo pine seedlings Fig. . Anet peMT and methanol represented the highest emission rates during heat exposure Fig. c and d. −1). This was in contrast to the cumulative MT emissions during the two heatwaves (DOY 114–131) which summed up to 28.1 ± 4.4SE mg (Table S2), and would hence account for a 19.7 ± 1.2SE % decrease of the total pool (as derived above). Considering specific compounds, we did not observe a significant impact of heat stress on any of the 14 MT measured (Table S2). Notably, the overall content of stress-induced SQT compounds (sum of 14) tended to increase compared to the control seedlings  and transpiration to reach zero between the two heatwaves .Monoterpene emissions are known to respond strongly to temperature increase by release from specific storage pools depending on diffusion parameters and volatility , similar to those of dried needle biomass of Scots pine (0.8%) reported by Vanhatalo et al. . UsuallyArabidopsis thaliana thermal pre-treatment (38 °C) induced salicylic acid accumulation (non-volatile form of MeSa) that caused thermotolerance of the plants at 47 °C . Mortality was more pronounced in the group of heat-drought-stressed seedlings, possibly due to damages from higher leaf temperatures caused by reduced evaporative cooling  and that shortly afterwards also dark respiration stopped, indicating that shoots became metabolically inactive. We found an increase of methanol emission in dying (but not in surviving) seedlings one day before this occurrence, while otherwise the emission rate was relatively stable as highlighted before Below is the link to the electronic supplementary material.


The principal objective behind the proposed design is to deal with the issues of intra‐patient parametric variation and non‐availability of all state variables for measurement. The proposed control technique for the T1D patient model is based on the attractive ellipsoid method (AEM). The observer and controller conditions are obtained in terms of linear matrix inequality (LMI), thus allowing to compute easily both the observer and controller gains. The closed‐loop response obtained using the designed controller avoids adverse situations of hypoglycemia and post‐prandial hyperglycemia under uncertain conditions. Further to validate the robustness of the design, closed‐loop simulations of random 200 virtual T1D patients considering parameters within the considered ranges are presented. The results indicate that hypoglycemia and post‐prandial hyperglycemia are significantly reduced in the presence of bounded ( Type 1 diabetes (T1D) patients reckon on multiple exogenous insulin infusions since their body is unable to secrete insulin (which is the primary regulator of glucose homeostasis), repercussion in prolonged elevated plasma glucose concentration (PGC). The extreme glucose excursions in both the direction from the euglycemic range of 70–180 mg/dl lead to hyperglycemia (PGC>180 mg/dl) and severe hypoglycemia (PGC<50 mg/dl) , 3 .The artificial pancreas (AP) system is essentially an automatic closed‐loop system for the exogenous insulin delivery via insulin pump as determined by a control algorithm based on the glucose measurements provided by the glucose sensors  . The isMathematical models that constitute the core of the model‐based control algorithms can be classified into intravenous and the subcutaneous T1D models, where the glucose measurements and insulin infusions are done intravenously or subcutaneously, respectively #  . In thiThe most significant issues in the domain of control of nonlinear systems are: (i) parametric uncertainty and modelling inaccuracy, (ii) presence of immeasurable states   and  H∞ controllers are often of high order and fragile in nature, (ii) SMC based techniques suffer from inherent chattering phenomenon that may result in aggressive exogenous insulin infusions leading to hypoglycemia.Most of the model‐based control design for diabetes patients are found to be based on PID control  , model  control  –25  and control  –28 . Th±30% parametric uncertainty and random meal disturbances.A robust observer based on AEM estimates the immeasurable states of the BMM in the presence of A robust state feedback control law is proposed for the BMM that provides optimal insulin dosage when model parameters are different for different subjects (intra‐patient variability).The observer gain and the controller gain are optimal as they are computed by solving a constrained optimisation problem with LMI conditions, thereby, ensuring analytical and numerical tractability.An output feedback control law was designed to (i) stabilise the uncertain nonlinear T1D system in the presence of exogenous meal disturbance and (ii) meet the required control performance by avoiding hypoglycemia.Finally, control variability grid analysis (CVGA) of 200 virtual T1D patients under the proposed observer based output feedback control law is performed for the evaluation of the efficacy as well as validation of the reliability of the proposed control technique.In context to the foregoing discussion on the existing methods for regulation of PGC in diabetes patients, a robust observer based output feedback controller for the nonlinear BMM utilising the analytic framework of AEM for a class of Lipschitz nonlinear system in the presence of time‐varying parametric uncertainty (intra‐patient variability) as well as exogenous meal disturbance is proposed for the first time. The theoretical contribution of the current work is the extension of the observer based output feedback stabilisation of nonlinear system which was presented in   to a roThe paper is organised into four major sections. Section 2 presents the mathematical model of the T1D patient, the design of the robust observer and the robust output feedback control law. The simulation results for different scenarios of the patients are presented in Section 3. Section 4 summarises the present work along with the scope of future extension of the work.Since BMM is a well known and popular T1D model that has been extensively studied and analysed in the literature, thus, the physiological intravenous BMM for T1D is presented briefly in the first Subsection.xi(t),i=1,…,3 represent the PGC (mg/dl), the delayed insulin action (min−1 ), the plasma insulin concentration (mU/L). Gb and Ib represent the basal (steady‐state) values of PGC and insulin concentration, respectively. The first ordinary differential equation (ODE) models the plasma glucose dynamics which tell us that the PGC is nonlinearly affected by the state x2(t) and the parameter c1 (min−1 ) denotes the insulin independent glucose utilisation factor (glucose effectiveness) [c3/c2 (L/(min×mU) ) stands for the insulin sensitivity in the second ODE that accounts for the delayed action of insulin on the PGC. The third ODE explains the insulin kinetics with the control input  u(t) appearing externally to it. The parameter c4 (min−1 ) stands for the insulin degradation rate [d(t) affects the PGC externally following a meal ingestion where c5 (min−1 ) is the rate of appearance of meal disturbance in the plasma glucose compartment.The BMM reported in , 26  is iveness)  . The raxd(t)=x1d(t)x2d(t)x3d(t)T is the deviated state about the equilibrium point x0=x10x20x30T=Gb0IbT=8007T of the system (ϕ(xd)=−x1d(t)x2d(t)00T, and B=001T, D=100T, C=100, xd∈ℜn, u(t)∈ℜm, y(t)∈ℜp, with n=3, m=1 and p=1 . The modern Artificial Pancreas System (APS) is equipped with glucose measuring devices that provide continuous glucose measurements periodically that is crucial for the controller design.A, B ) and  are found to be controllable and observable for the considered model.The pair  represents the uncertain part of the system matrix, where the elements vary within a plausible range of ci,i=1,…,4 as mentioned in Table X+>0,D+>0andδ>0 are known apriori and xdmax represents the maximum deviation of xd(t) . The elements of the uncertain matrix, ΔAt vary within an interval as provided in Table ΔAt is the worst case norm of the uncertain matrix calculated using the parametric variations as given in Table Considering the range of parameters placed in Table x˙d(t)=AL ensures the convergence of the state estimation error, (ii) Stage 2: compute state feedback gain with parametric uncertainties ensuring the convergence of the states and finally (iii) Stage 3: design of the output feedback control law using the estimated state from the observer and the state feedback gain from the controller.The design philosophy of the current research work is illustrated in Fig. The potential advantage of this method is that it does not require to test the controllability and observability of the nonlinear system, observer and controller gains can be computed separately and more importantly the implementation is output feedback law and not the state feedback law.Following definition and Lemmas will be useful to design an observer for the system   in an LE(Pattr) . It is said to be an attractive ellipsoid for the trajectory eo(t),t>0 if it tends asymptotically inside this ellipse. Mathematically, it can be expressed as: limsupt→∞eoTtPattreot≤1.  Let us Φ(xd) is called Lipschitz function, if it satisfies,(xd(t),x^d(t))∈ℜn and Lϕ>0 in (Schur compliement lemma:): [G, H and I, GHHTR is equivalent tolemma:):   For anye system   in presIn this subsection, a robust observer is designed for a class of Lipschitz nonlinear uncertain system   adoptinL is the observer gain matrix, y(t)=Cxd(t)=x1d(t) is output of the uncertain T1DM model DM model   with C=ues from   and (10DM modellemma in  . The obPattr=α/(ε(δ2X+2+D+2))P, indicating that the estimation error converges to an attractive ellipsoid as defined in Definition 1.P=PT>0 . Differentiating on both sidesLet us consider a positive definite storage function,tituting   in (19 Further   can be εIn×n and αV(eo) on the left side of the above expression  applied to Pc, indicating that the state xd(t) converges to an attractive ellipsoid as defined in Definition 1.εcξ2(t) and αcVc(xd(t)) on the right side of =ΛoPcPc−εcIn×n . Notice that Wc is a BMI. Introducing a appropriate non‐singular transformation, Tc=Pc−100In×n one can easily showLet us consider a positive definite quadratic storage functionVcxd(t):ntiating   one caniating  and carrying out the operation as mentioned in (Λm=(A+BK+αc/2In×n)Pc−1+Pc−1((A+BK+αc/2In×n) ⊺+εcLϕ2In×nPc−2, one can writePc−2 can be estimated by the following inequality, Pc−2<Q . Applying Schur complement lemma in  the term Λm in (Λm=Λ11c as defined in Theorem 1 and (ii) substituting these new variables, X=Pc−1 and Yc=KPc−1 in  is computed by reformulating the above theorem as a minimization problem. The result is presented in the form of corollary below.Pattrc−1=X is minimised such that the closed‐loop states of  . The closed‐loop dynamics by substituting the following output feedback control law:−2PBKeo(t) ’. Since Theorem (4) guarantees the convergence of the state estimation error eo(t), the term ‘ −2PBKeo(t) ’ can be neglected. Hence, the state vector xd(t) converges to an attractive ellipsoid.The objective of this sub‐section is to design an output feedback control law such that the uncertain system in   with th)Using  , one caction in   and difUsing (x˙d(t)=( same as   except x1>50mg/dl ), (ii) prolonged post‐prandial hyperglycemia in the presence of external meal disturbance is avoided and (iii) the control signal should be non‐negative.This section presents the closed‐loop simulation results of the designed controller on the T1D subjects with time‐varying uncertain model parameters and uncertain external meal disturbances such that (i) PGC must not fall below the severe hypoglycemic level  whose lower limits are always non‐negative. The Lipschitz constant is computed by considering the above bounds on the states.The numerical values for and δ in   are conThe optimal values for the controller gain matrix, K∗ in   is compK∗ in  , the coix L∗ in   and theProtocol: In this scenario, the objective is to investigate the controller's ability to bring PGC to normal value from the hyperglycemic state within t=150min in the presence of a high initial meal disturbance. The initial conditions describing the physiological conditions of the T1D patients are x10=200mg/dl, x20=0.001min−1 and x30=7mU/l.Protocol: A simulation scenario of 3 days (4320 min) is carried out here, the parameters of the system  ) is brought to the basal value Gb=80mg/dl successfully by the designed controller in presence of time‐varying uncertainty thus avoiding any instance of hypoglycemia. Further, from Fig. t=150min . The control input (insulin infusion rate) determined by the designed controller is depicted in Fig. c3c2 as depicted in Fig. The performance of the robust observer is depicted in Fig. ci,i=2,…,5 chosen from the uncertainty range specified in Table Three virtual T1D patients referred as nominal, maximum and minimum takes three different set of values of B, 6% of the dots lie in Grid Lower D and 1% are in Grids Lower B and Lower C, respectively. So, it can be concluded that the proposed control technique completely avoids hypoglycemia (BGC<50 mg/dl) thus validating its effectiveness in dealing with the intra‐patient variability. Further, long‐term implications of post‐prandial hyperglycemia is also significantly reduced by the proposed method.Control variability grid analysis (CVGA)   is perfx1 below 180 mg/dl, tx1<180=200min, and ultimately to the basal value, Gb and the time taken to bring x1 to Gb, tx1=Gb=700min . As illustrated in Fig. umax=60mU/l/min . Such aggressive control action can be dangerous due to hypoglycemia if the PGC of the T1D patient is at lower values. As depicted in Fig. d(t), unlike the present work as mentioned in Table A comparative analysis of the closed‐loop performance of the proposed AEM observer based controller with single order sliding mode control (SOSMC)   and higA simple yet effective robust observer based output feedback control technique based on the attractive ellipsoid method has been proposed for an uncertain Bergman's minimal model. The CVGA plot clearly reveals the effectiveness and reliability of the proposed technique in regulating the glucose concentration and insulin infusion near to normal subjects under parametric uncertainty and random meal intake. Severe hypoglycemic events which are the primary concern of Artificial Pancreas is eliminated successfully. The future scope of the proposed robust observer based control technique based on the attractive ellipsoid method can be the extension of the robust control philosophy to the more complicated subcutaneous T1DM model that finds an immediate application in the Artificial Pancreas System.The authors thankfully acknowledge the support of Prof. Alexander Poznyak, CINVESTAV‐IPN, Mexico D.F. for his constructive technical inputs towards completing the work.


A superior analgesic method in perioperative pain-management of patients receiving total knee arthroplasty is the subject of controversial debate. Although higher cost-efficiency is claimed for the local infiltration analgesia (LIA), there is a lack of data on its costs compared to peripheral nerve block anaesthesia (PNBA). The goal of this study was to investigate the differences in immediate perioperative costs between the LIA and PNBA in treatment of patients receiving total knee arthroplasty.The comparison was conducted based on a randomized controlled clinical trial examining 40 patients with elective, primary total knee arthroplasty . The analysis included surgical case costs, anaesthesiological case costs, material, costs of postoperative opioid requirements and catheter review visits for patients receiving PNBA.p = 0.851). While there was no statistically significant difference in surgical case costs, the anaesthesiological costs were lower with the LIA procedure . Material costs in the LIA group were 4.18€/patient and 94.64€/patient with the PNBA. Costs for postoperative opioid requirements showed no statistically significant difference between the two procedures.The overall mean costs for the LIA-group were 4328.72€ and 4368.12€ for the PNBA  and registered with data safety authorities. Study patients provided written informed consent to participate in the trial. Study registry: ClinicalTrials.gov,  The local infiltration analgesia (LIA) and peripheral nerve block anaesthesia (PNBA) are techniques in the postoperative pain-management of patients undergoing major orthopaedic surgery such as total knee replacement.PNBA – a regional anaesthesia procedure with/without catheters - is seen as an effective method in postoperative pain-management of endoprosthesis implantation . The LIAHowever, research-based discussion on the preferred method remains controversial , 5. SomeDue to this ambivalent data, there is a rising interest in economic analyses complementing clinical and more patient-orientated aspects of determining which procedure is preferable . PreviouClinicalTrials.gov, NCT03114306) [The study is a secondary analysis of a randomised controlled trial (RCT) published by Kastelik et al. (study registry: 3114306) . The bac3114306) .The main subject of the secondary analysis at hand was the difference in overall case costs between LIA and PNBA procedures.All patients received postoperative analgesia following a standardised protocol of opioids , dipyrone and non-steroidal anti-inflammatory drugs . For additional analgesic treatment in case of acute pain scores on the numeric rating scale (NRS) > 6, patients received 10 mg of oral morphine.During the LIA procedure the surgeon injects 150 ml Ropivacain 0.2% into the periarticular structures, thereby blocking the sensitive nerve endings of the knee. The targeted structures are the subcutaneous and periarticular soft tissues as well as the joint capsule. LIA is often administered during the orthopaedic implantation of endoprosthesis, immediately before suture .For PNBA, ultrasound-guided sub-gluteal block of the sciatic nerve as well as the adductor canal block with anaesthesia of the saphenous nerve were performed in the primary study . Both neTo calculate the overall case costs for each procedure we summed up the surgical case costs, anaesthesiological case costs, postoperative opioid requirements, material costs and costs of catheter review visits (only in PNBA procedure). The specification for the times used for the calculations was taken from Bauer et al. on perioperative process times and indicators  and is sSurgical case costs were made up of the price per minute for personnel , operational infrastructure and material (33.77€) and were multiplied by the incision to suture time (O8-O10 ). The exAnaesthesiological case costs consisted of the price per minute for personnel (doctor and anaesthesiology nurse), infrastructure and material (10.28€) and were multiplied by the total time of anaesthesia (beginning of the anaesthesiologist’s presence with the patient until end of patient monitoring by the anaesthesiologist in the operating room (OR) or similar (A5-A9) ).Material costs Tables  and 2 weOpioid costs for the treatment of acute postoperative pain included the individual and average dosages of tilidine/naloxone retard and oral morphine 10 mg tablets [Catheter review-visits for patients receiving the PNBA were calculated with 6.25€ per visit based on the assumption of a before-tax income of 25€/h of an anaesthesiological nurse and an average time per visit of 15 min.Total costs were obtained by the summation of the above variables. All costs and prices were taken from the expenses of the Charité–Universitätsmedizin Berlin at the time the study was conducted.The statistical analysis of our data was conducted in IBM SPSS 25. Descriptive data was summarised by mean and standard deviation or median depending on statistical distribution. The t-test for independent samples was used to test for statistical significance of parametric data. Data for opioid requirement for postoperative acute pain as well as for induction costs were analysed using the Mann-Whitney-U-Test. For all statistical analysis a 5% two-sided alpha level was applied. A case number analysis was not conducted for this secondary end-point as this was a descriptive non-confirmatory supporting analysis. The statistical graphic was originally produced with SPSS 25 revised as vector graphics using Affinity Designer 1.8.6. Images were designed to be accessible for people with anomalous trichromacy, dichromacy and monochromacy.p = 0.132).Mean surgical case costs within the OR for the LIA procedure were 2933.81€ (SD ± 463.94€). For the PNBA mean costs were 2690.67€ (SD ± 532.67€). There was no statistically significant difference between the two procedures (p = 0.048).Mean anaesthesiological case costs for the LIA were 1370.26€ (SD ± 209.54€) and 1542.45€ (SD ± 313.12€) for the PNBA. This difference was statistically significant . Mean costs of cumulative postoperative oral morphine per patient were 1.46€ (SD ± 2.08€) for LIA and 1.59€ for PNBA (SD ± 2.08€) (p = 0.646).There was no statistically significant difference in the cumulative use of postoperative opioids. Mean costs of cumulative postoperative tilidine/naloxone per patient were 19.01€ (SD ± 2.44€) for LIA and 17.22€ (SD ± 4.85€) for PNBA (Mean costs for catheter review-visits amounted to 21.56€ per patient (SD ± 4.74€) and were added to the PNBA-group only, as outlined above.p = 0.851). Figure Overall, the mean costs for the LIA procedure were 4328.72€ SD ± 644.14€) per patient compared to 4368.12€ (SD ± 677.24€) for the PNBA procedure. This difference was not statistically significant (4€ per pap = .758) [The authors of the primary study reported no significant postoperative complications that may have had an impact on costs with either procedure and there was no significant difference in total hospital length of stay between the two groups ( = .758) . There wOur analysis shows that there seems to be no statistically significant difference in immediate perioperative costs between the two analgesic procedures in our hospital. Indeed, both concepts seem to provide fast-track mobilisation and allowed early in-hospital recovery after surgery and ambulation maintaining high patient comfort with comparable costs (patient-related outcome measure).Although the results show that the anaesthesiological case costs were significantly lower for the LIA procedure, this was primarily due to the prolonged anaesthesia induction time for the PNBA  and willKastelik et al. reported a longer mean incision to suture time in the LIA group  which, aMaterial costs were considerably lower in the LIA group, which was consistent with existing research . Other tOne objective of the development of the LIA was to reduce risk of postoperative complications such as venous thrombosis or nosocomial infection  achievedMobilisation to stand was successfully achieved in the recovery room in 90% of LIA and 95% of PNBA patients in the primary study . This maN = 40), which was powered for the primary endpoint of mobilization. Thus it may be insufficient to draw conclusive evidence towards economical differences between the two procedures without further research. However, due to the randomization in the study design risk of confounding bias stays relatively small. Further research should expand on these issues and potentially include other postoperative costs such as physical rehabilitation and overall hospital length of stay.Limitations of our work may include the restricted transferability of cost structures of a university hospital like the Charité to other institutions on a national and international level. The surgical case costs as the largest cost factor may differ depending on the surgeon’s experience in the handling of the LIA and the overall procedure of TKA. Another limitation may be the relatively small sample size (This study expands on the financial evaluation for determining a preferred procedure in perioperative pain management for patients receiving TKA. Although we were able to describe in detail lower anaesthesiological case costs with the LIA, there was no difference found between the overall case costs for LIA and PNBA. Therefore, based on our analysis we can conclude that the organisational structure and process optimization are the key to reduce costs for either procedure.It should be noted that the determination of an analgesic procedure for patients receiving knee replacement surgery should always rely primarily on medical indications and should therefore be chosen by carefully evaluating individual risk factors and benefits for each individual patient. Economic factors should only be seen as secondary arguments for the individual but can play a considerable role in the effective allocation of resources and organisational planning.


The main goal of this work was designing a single qubit neural quantum circuit for performing Exclusive-OR, a concrete example of an operation requiring multiple layers in classical neural networks. The corresponding Qiskit code was tested on both simulators and IBM's “ibmqx2” five-qubit quantum processor. Further analyses along training the proposed neural quantum circuit are currently in progress.• A single qubit neural quantum circuit for performing XOR was tested on IBM Q Experience• XOR is a concrete example of an operation requiring multiple layers in classical neural networks  Specifications tablen input values “connected” to a single output. Each input channel is characterized by a weight parameter. The output is calculated using the activation function:y is the output, x the input vector, w the trainable weight vector, and t a threshold trainable parameter.A perceptron Although of high interest in both neurosciences and artificial intelligence, the model was proven to be limited to linearly separable functions In the field of Quantum Neural Networks there are various attempts θ + s and θ – s, where θ represents the circuit parameters, and s is a macroscopic shift. Thus, the circuit could be trained using gradient descent methods, such as backpropagation. The detailed method, including PyTorch code examples, is also discussed in Ref. Hybrid quantum-classical neural networks are described in The ability of single quantum neuron to compute linearly non-separable functions is directly connected with the basic property of quantum mechanics that a wave function is invariant under application of global phase RY(θ) gate, where θ is a trainable parameter. The output in z-basis is given by the expectation:i)pRZRX is the input vector,  are the “dendrites” trainable weights, and α is also a trainable parameter. The Hadamard gate is used for initializing the qubit to the |+> diagonal state, while the angles of the two rotation gates are controlled by the  input vector. The output is obtained by collecting the measurement statistics for calculating the expectation σ . The co1= θ2=π,θ for α = -π/2. This case is illustrated, for each input vector, in (The circuit could be trained using gradient descent methods as it was designed following the parametrized quantum circuits principles described in ctor, in .Fig. 2SiThe circuit was implemented on IBM Quantum Composer A particular single qubit parametrized quantum circuit for performing XOR was successfully tested on both simulators and the “ibmqx2” five-qubit quantum processor. Further analyses along training the proposed neural quantum circuit are currently in progress.Co-submissions are papers that have been submitted alongside an original research paper accepted for publication by another Elsevier journalThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The author declares no conflict of interest.


Conventional ratiometric luminescence thermometry often relies on thermal coupling between two closely lying energy levels governed by Boltzmann’s law. Despite its simplicity, Boltzmann thermometry with two excited levels allows precise temperature sensing, but only within a limited temperature range. While low temperatures slow down the nonradiative transitions required to generate a measurable population in the higher excitation level, temperatures that are too high favour equalized populations of the two excited levels, at the expense of low relative thermal sensitivity. In this work, we extend the concept of Boltzmann thermometry to more than two excited levels and provide quantitative guidelines that link the choice of energy gaps between multiple excited states to the performance in different temperature windows. By this approach, it is possible to retain the high relative sensitivity and precision of the temperature measurement over a wide temperature range within the same system. We demonstrate this concept using YAl3(BO3)4 (YAB):Pr3+, Gd3+ with an excited 6PJ crystal field and spin-orbit split levels of Gd3+ in the UV range to avoid a thermal black body background even at the highest temperatures. This phosphor is easily excitable with inexpensive and powerful blue LEDs at 450 nm. Zero-background luminescence thermometry is realized by using blue-to-UV energy transfer upconversion with the Pr3+−Gd3+ couple upon excitation in the visible range. This method allows us to cover a temperature window between 30 and 800 K.Ratiometric luminescence thermometry with trivalent lanthanide ions and their 4f According to the Stefan−Boltzmann law, the intensity of the emitted radiation scales with the fourth power of temperature and is expected to be sensitive for high temperatures and objects with high emissivity.Chemical reactions, biological functionalities, and various physical phenomena are all strongly determined by the local temperature. The measurement of local temperatures on successively smaller scales requires new methods of temperature sensing that rely on a remote detection principle. Several such concepts already exist. The most commonly known example is infrared (IR) thermography or pyrometry6. It requires a simple setup consisting of a laser excitation source, luminescent micro- or nanocrystals or molecules embedded in the object of interest, and a fast and efficient light detection system9. Its promising advantages over thermography include the lower accessible temperature ranges and non-invasive temperature detection below surfaces. Its successful application in biological media has been highlighted for many different optical centres, such as nitrogen vacancy (NV−) centres in fluorescent nanodiamonds12, semiconducting quantum dots (QDs)15, bulk halides17, and organic fluorophores21.An alternative method of remote temperature sensing is luminescence thermometry, which exploits the fact that luminescence properties such as the emission intensity or the luminescence decay time are highly dependent on the local temperature of the surroundings of the luminescent species23. Their rich electronic level structure that arises from the partly filled 4fn (n = 1–14) inner shell with characteristic energy gaps on the order of thermal energies  allows for thermal coupling between energetically adjacent levels. Most representatives of this class of luminescent thermometers thus rely on ratiometric sensing; in ratiometric sensing, the temperature is measured by means of the intensity ratio of the emission bands from two thermally coupled excited levels. The key advantages of this approach are its simplicity and that it is self-referenced. Moreover, the narrow linewidths of the intraconfigurational 4fn−4fn transitions minimize spectral overlaps between emission lines from two thermally coupled energy levels, even when the energy separation is small. The approximate independence of their energies from the surrounding host material related to the low radial extension of the 4f orbitals allows us to select the emission energy range of interest by appropriately choosing the lanthanide ion. Recently, there has also been progress in reverting this scheme and performing thermometry by detecting a reference emission from the same excited level upon dual excitation from two thermally coupled ground levels26. Luminescence (nano)thermometry has been demonstrated to be both a precise and accurate technique for probing fundamental thermodynamic phenomena at the micro- and nanoscale29 and helps to assess the local temperature fluctuations in tissue35 or in chemical reactors40. Currently, there has been observable progress on how to implement and standardize this technique for applications45, the connected limitations47, or how to couple temperature sensing with other functionalities such as single-molecule magnetism49 and (meso-)porous materials51, e.g., theranostics52.An alternative emerging class of luminescent thermometers are micro- or nanocrystals doped with trivalent lanthanide ions54. It has been recently demonstrated that despite their appealing simplicity, Boltzmann-based thermometers with two excited levels suffer from a fundamental thermodynamic limitation55. At very low temperatures compared to the energy gap ∆E21 between the two excited levels |1〉 and |2〉 (kBT ≪ ∆E21), the probability of thermally exciting the population from the lower to the higher excited emissive level is vanishingly small, which translates to a negligible intensity of the emission from the higher excited level. In addition, the nonradiative absorption rate governing the thermalization from level |1〉 to |2〉 becomes so slow that it can no longer compete with the radiative decay of level |1〉, which leads to a decoupling of the two excited states. In contrast, at very high temperatures compared to the energy gap (kBT ≫ ∆E21), the populations of the two excited levels in the thermodynamic limit effectively equalize and thus lower the sensitivity of the thermometer, which relies on a relative net change in population between the two excited levels. Both described extremes diminish the overall achievable precision of the thermometer and imply that any Boltzmann-based two-level thermometer only performs with sufficient statistical precision within a limited temperature range dependent on the energy gap ∆E21 between the two excited levels.The intensity ratio of the emission lines originating from two thermally coupled excited levels within the same configuration follows simple Boltzmann statistics if thermalization is faster than population decay (by any radiative or nonradiative pathways)58, no clear guidelines have been established with regard to the most advisable temperature ranges for energy gaps for two or more higher excited levels thus far. Knowledge of the fundamental thermodynamic properties of such a thermally coupled multilevel Boltzmann-based luminescent thermometer helps decide at which temperatures a change to another luminescence intensity ratio (LIR) is advisable to achieve the highest thermometry precision and which combination of energy gaps is optimal for the (wide) temperature range of interest.A way to circumvent this thermodynamic limitation and to widen the temperature range for the highest precision of a Boltzmann-based thermometer is to extend the concept of thermal coupling between more than two energetically close excited levels. This method allows us to retain both an optimized response and sensitivity of a single luminescent thermometer by monitoring emission intensity ratios from multiple higher energy excited levels separated by different energy gaps from the lowest excited emissive state. While this has already been realized on a qualitative levelλmaxT = b) with b ≈ 2.8978 mm∙K) states that the peak wavelength of the Planckian black-body spectrum shifts to shorter wavelengths at high temperatures. While the peak maximum clearly lies in the infrared range at usual lab-accessible temperatures (T < 2000 K), the short-wavelength tail of the black-body spectrum can already interfere with the luminescence spectrum in the visible range at temperatures as low as 800 K. Thus, any ratiometric luminescent thermometer devised for high temperature sensing best relies on emission in the ultraviolet range, which is clearly unaffected by black-body background at temperatures below 1200 K.Luminescence thermometry at high temperatures also suffers from another practical problem. Any solid emits thermal or black-body radiation, which becomes more intense at high temperatures. Moreover, Wien’s displacement law  crystal fields and spin-orbit levels of the UV B-emitting lanthanide ion Gd3+; we were motivated by thermodynamic considerations and demonstrated how a single luminescent phosphor can be optimized for precise thermometry from cryogenic (30 K) to high temperatures (800 K) with constantly high relative sensitivities above 0.5% K−1. Intense excitation with a cost-effective blue wavelength of 450 nm is interesting for practical applications. This is possible by co-doping with Pr3+ in huntite-type crystallizing YAl3(BO3)4 (YAB)59 and upconversion into its 4f15d1 configuration64, followed by efficient energy transfer to the 6IJ’ (J’ = 17/2…7/2) levels of Gd3+. Not only does this offer the possibility of background free upconversion thermometry, but it also bears potential for applications such as UV lasing64 or visible light excited photocatalysis65 combined with local temperature sensing. Similar visible-to-UV upconversion relying on triplet-triplet annihilation has otherwise been recently reported by Harada et al. for metal-organic compounds66In this work, we present such a designed multilevel luminescent thermometer that uses the three excited Sa(T), of the luminescence intensity ratio (LIR) is directly related to the absolute effective thermal population of the microstate of the higher excited level from which the emission arises. The relative sensitivity, Sr(T), in contrast, relates to the relative net change in population between the two thermally coupled excited levels of interest55. As indicated above, the thermodynamically optimized performance of a two-excited level thermometer is limited to a small temperature range only. This performance is related to the low response at low temperatures due to the almost negligible thermal population in the higher excited state and a low relative sensitivity at high temperatures due to almost equalized populations (per microstate) in both thermally coupled excited levels. The optimum temperature range depends on the probed energy gap ∆E21 and is given by55The benefit of a third emissive level on the performance of a luminescent thermometer can be appreciated as follows. The thermal response , E31 = (1 + s)∆E21 from the lowest excited level |1〉. According to Eq. , Eq.  into the 4f15d1 configuration of Pr3+ at energies in the range of 40,000 cm−1 can take place72, which is followed by an energy transfer to the 6IJ’ levels of Gd3+ at energies of approximately 37000 cm−1 (270 nm). The dependence of the Gd3+-related emission intensity on the incident power of the OPO also indicates a two-photon upconversion process  levels and the 8S7/2 ground level of Gd3+, which does not have any intermediate electronic levels, as well as the high mutual lanthanide distances in the YAB host structure (~ 5.9 Å)59, allow an increase in the Gd3+ concentration to 20 mol% without any observable concentration quenching effects , normalized to its value at the highest measured reference temperature, T0, thus follows the law74kB is the Boltzmann constant and ∆Em1 (m > 1) is the energy gap between the two thermally coupled levels and is the only fitting parameter. A fit to a linearized version of Eq. (ECF = (69 ± 5) cm−1, in excellent agreement with the value of 72 cm−1 determined from the emission spectra. It should be noted that a corresponding splitting of the 8S7/2 level into Kramers’ doublets is much smaller (on the order of only 1 cm−1) if spin-orbit coupling is only intermediate because of the lack of orbital degeneracy given the quantum number L = 075. This makes the 8S7/2 ground level an effective single level in the case of Gd3+.Boltzmann thermometry with Gdsee Fig. . These tsee Fig. , whose i77gm as the degeneracy of the higher excited level (gm = 2 for Kramers’ doublets), knr(0) as an intrinsic nonradiative rate governed by the properties of the electronic transition, andp as the number of phonons consumed during the thermalization between the two excited levels (∆Em1 = p = 1). This temperature-dependent nonradiative absorption rate competes with the radiative decay  rates from level |1〉. At sufficiently low temperatures, the nonradiative absorption rate decreases than the intrinsic radiative decay rate, and thus, Boltzmann thermalization is kinetically inhibited. We have recently shown that a very accurate estimate for the expected onset temperature of Boltzmann behaviour, Ton, is given by55The LIR deviates from the expected Boltzmann behaviour at temperatures below 25 K. This observation is related to a kinetic limitation. The nonradiative absorption rate from the lower to the higher excited level, ECF = 72 cm−1, the expected onset temperature for Boltzmann thermalization between the two excited levels is thus approximately 23 K, very close to the observed deviation from Boltzmann behaviour below 25 K. A similar deviation has also been observed in the case of Cr3+78, which can be explained by the same kinetic effect. In turn, the optimum temperature range for the most precise thermometry that exploits this energy gap between the two Kramers’ doublets of the 6P7/2 level of Gd3+ is between 30 and 51 K according to Eq. (Sr(T), as defined bySr(30 K) = 11.6% K−1 and Sr(51 K) = 3.98% K−1 in this optimum range, which is clearly above the usually desired threshold of Sr = 1% K−1 in practice23. In fact, the relative sensitivity at 30 K is the highest reported value for luminescent cryothermometers at that particular temperature thus far80. It is important to note, however, that it is both the balance between reliably detectable luminescence signals and relative sensitivity that determine the overall precision of a luminescent thermometer 55,81. Another important point is that for accurate cryothermometry with small energy gaps, a high spectral resolution spectrometer is also required to separate two emission lines that are close in wavelength. Finally, that high relative sensitivities at cryogenic temperatures are not surprising, as can already be appreciated from the T−2 dependence in Eq.  cm−1, again in excellent agreement with the value of 506 cm−1 that was spectroscopically deduced from the emission spectra. With this energy gap, the most suitable temperature range for high-precision luminescence thermometry is between 213 and 364 K with corresponding relative sensitivities of Sr(213 K) = 1.60% K−1 and Sr(364 K) = 0.55% K−1. These relative sensitivities are almost one order of magnitude higher than the corresponding sensitivities for thermometry with crystal field splitting of 72 cm−1 in this temperature range. This result demonstrates that the concept of multiple excited states with different energy separations could realize high-temperature sensing accuracy over a wider temperature range, given sufficiently high photon counts of the emission bands of interest. As in the case of cryothermometry, the LIR deviates from Boltzmann behaviour below 200 K, which is a consequence of the very slow nonradiative absorption in that temperature range55. This observation also coincides very well with the estimated onset temperature for Boltzmann behaviour at Ton = 163 K according to Eq.  cm−1, again in excellent agreement with the spectroscopically determined value of 1120 cm−1. This energy gap is optimally suited to measure temperatures between 472 and 805 K with corresponding relative sensitivities of Sr(472 K) = 0.73% K−1 and Sr(805 K) = 0.25% K−1, which is an improvement by a factor of 2.2 compared to thermometry with the 6P5/2−6P7/2 gap in this temperature range. Here, again, thermalization and the sustainment of a Boltzmann equilibrium between the 6P3/2 and 6P7/2 spin-orbit levels of Gd3+ is only observed above temperatures of at least 450 K, while Eq.  or whether continuous exploitation of the LIR between the transitions from the 6P5/2 and 6P7/2 levels up to higher temperatures is more favourable based on the higher luminescence intensity of the 6P5/2 → 8S7/2 radiative transition. For that purpose, we calculated the minimum theoretical relative temperature uncertainty for both thermometry measures within the optimum temperature range of the 6P3/2−6P7/2 gap (472−805 K). The relative temperature precision of a ratiometric Boltzmann-based thermometer is given by55I10 denotes the intensity (in integrated photon counts) of the lower energetic emission and I10 = 107 counts of the 6P7/2 → 8S7/2-based emission, the LIR between the radiative transitions from 6P5/2 and 6P7/2 still gives rise to a lower relative temperature uncertainty (ca. 0.08%) based on the higher relative intensity of the 6P5/2 → 8S7/2 transition than the corresponding LIR involving the radiative transition from the higher energetic 6P3/2 level (relative temperature uncertainty of ca. 0.2%). In that case, it is advisable to still retain the LIR stemming from the adjacent 6P5/2 and 6P7/2 levels. Thus, it depends on the trade-off between practically achievable intensities and thermodynamically guided optimum temperature range, which is the more advisable choice as a temperature measure to probe high temperatures with Gd3+ in YAB: Pr3+, Gd3+. Irrespective of the choice, it is possible to keep the absolute temperature uncertainty below a threshold of 1.3 K even at 820 K. We do stress, however, that these relative temperature uncertainties are purely statistical and do not include any additional systematic error that could occur in a real-case application. It is also important that these low uncertainties require a high integrated intensity (~107 cts) of the 6P7/2 → 8S7/2-related emission of Gd3+, which may be difficult to achieve in an application based on the proposed upconversion.The photoluminescence spectra reveal that the intensity of the radiative emission from the 3+, which exhibits three energetically well-isolated 6PJ  levels in the UV range that can additionally rely on splitting in a local crystal field. Gd3+ is thus a prime choice for especially high-temperature ratiometric luminescence thermometry, as the black-body background does not interfere in this emission range. Moreover, it absorbs and emits in the solar-blind spectral range, and thus, any background signal from ambient sunlight is also avoided. Since Gd3+ is a weak absorber, we demonstrated an alternative excitation scheme with a cost-effective and well-established excitation wavelength of 450 nm available from blue LEDs via Pr3+ in a blue-to-UV upconversion process. By ground state absorption into the 3P2 level, followed by efficient excited-state absorption into the 4f15d1 configuration of Pr3+, it is possible to indirectly excite Gd3+ by energy transfer into its 6IJ’ (J’ = 17/2…7/2) levels, from which the excited state population finally undergoes nonradiative relaxation into the 6PJ levels. Optimized Boltzmann cryothermometry between 30 and 50 K was possible with two crystal field components of the excited 6P7/2 level separated by 72 cm−1. Temperature sensing in the range of room temperature is better performed by exploiting the LIR of the radiative transitions stemming from the 6P5/2 and 6P7/2 spin-orbit levels of Gd3+ separated by 506 cm−1. Finally, high-temperature thermometry between 470 and 800 K is also possible using Boltzmann thermalization between the 6P3/2 and 6P7/2 levels of Gd3+, with an energy separation of 1120 cm−1. Both the 6P3/2 → 8S7/2- and the 6P5/2 → 8S7/2-based emissions were compared in terms of the overall achievable temperature precision of the respective ratiometric thermometry approaches. While the 6P3/2 → 8S7/2-based emission is very low in intensity, it offers the necessary high relative sensitivity at high temperatures. It was shown that the final choice for high-temperature thermometry with Gd3+ depends on the practically achievable integrated intensity of the lowest energetic 6P7/2 → 8S7/2 transition.Single ion luminescence thermometry based on a Boltzmann equilibrium between two emitting excited levels is both thermodynamically and kinetically limited to work with optimized precision only within a certain temperature range. If a luminescent thermometer is supposed to cover wider temperature ranges, thermal coupling between more than two excited states must be exploited. In this work, we offer quantitative guidelines based on a recently developed theoretical framework that allows us to extend the usability of a Boltzmann-based luminescent thermometer over wider temperature ranges by carefully choosing the energy gaps between multiple thermally coupled levels. We demonstrate the concept practically for the case of Gd3+, Gd3+-coactivated YAl3(BO3)4 phosphor with Gd3+ as a Boltzmann-based single ion luminescent thermometer. UV upconversion emission from the 6PJ levels of Gd3+ is sensitized via two-step excitation of Pr3+ at 450 nm to the states of the 4f15d1 configuration, followed by energy transfer to Gd3+, which is practically feasible with the use of high-power blue LEDs. Analysis of the temperature-dependent emission demonstrates how simple thermodynamic principles can be applied to circumvent the fundamental limitations in temperature precision of a conventional two-level single ion luminescent thermometer to finally make one thermometry system applicable to a wide range of temperatures while retaining high precision. This work paves the way for the targeted design of luminescent thermometers tailored towards applicational requirements for temperature accuracy within specific temperature ranges.In summary, a theoretical framework is presented to quantitatively understand and design single ion thermometers relying on multiple thermally coupled excited states to widen the window for accurate thermal sensing. The concept is shown to be feasible using a Pr3)3 ∙ 6 H2O , Al(NO3)3 ∙ 9 H2O , Pr(NO3)3 ∙ 6 H2O , Gd(NO3)3 ∙ 6 H2O , H3BO3  and urea  were used without further purification.Y were prepared by a modified urea–nitrate solution-based combustion route83. Stoichiometric amounts of Ln(NO3)3 ∙ 6 H2O  and Al(NO3)3 ∙ 9 H2O were dissolved in distilled H2O (approximately 40 ml) under vigorous stirring at room temperature. Subsequently, solid urea (molar ratio urea/lanthanide ions = 3:1) and H3BO3 (5 mol% excess) were added to the transparent nitrate-containing solution. The solution was heated to 80 °C to improve the solubility of the H3BO3 and constantly stirred for 30 min at that temperature. The beaker was covered with parafilm to prevent excessive solvent evaporation. The resulting solution was quickly transferred to an alumina crucible and placed into a preheated furnace at 500 °C in air for 10 min to initiate combustion. The thus-formed colourless solid precursor was carefully ground with additional solid H3BO3  in a mortar to account for the losses that occurred during the combustion step. The solid mixture was finally sintered at 1100 °C for 6 h in air. After naturally cooling to room temperature, the obtained colourless residue was ground to a fine powder, and its phase purity was verified by powder X-ray diffraction , U = 40 kV, I = 20 mA, reflection mode). The X-ray diffraction pattern was scanned in a 2θ range between 10° and 80° with a step size of 0.02°  was employed as an excitation source.Excitation was performed with an external pulsed wavelength tuneable Opotek Opolette HE 355 II  OPO pumped by a frequency-tripled Nd:YAG laser at a repetition rate of 20 Hz and a temporal pulse width of approximately 6 ns. Emission spectra were acquired on an Edinburgh FLS920 spectrofluorometer  equipped with a 0.25 m double Littrow-configuration grating monochromator blazed at 300 nm and a Hamamatsu R928  photomultiplier tube (PMT) for photon detection. All emission spectra were corrected for grating efficiency and detector sensitivity. Temperature-dependent measurements below room temperature were performed with an Oxford Instruments liquid He flow cryostat  and an external temperature control unit, which measured the temperature by means of a thermocouple in direct contact with the powder sample holder. High-temperature luminescence spectra were acquired by placing the sample into an externally water-cooled Linkam  THMS600 microscope stage (±0.1 °C temperature stability). The temperature was externally controlled by a thermocouple in immediate contact with the sample holder. Photoluminescence decay curves were acquired by pulsed excitation with the OPO and detection of the time-resolved signal with a multichannel scaler (MCS) attached to a Hamamatsu H7422 PMT  for minimized background. For the demonstration of the excitation of the upconverted emission of GdSupplemental Material


Glioma is a classical type of primary brain tumors that is most common seen in adults, and its high heterogeneity used to be a reference standard for subgroup classification. Glioma has been diagnosed based on histopathology, grade, and molecular markers including IDH mutation, chromosome 1p/19q loss, and H3K27M mutation. This subgroup classification cannot fully meet the current needs of clinicians and researchers. We, therefore, present a new subgroup classification for glioma based on the expression levels of Gβ and Gγ genes to complement studies on glioma and Gβγ subunits, and to support clinicians to assess a patient’s tumor status.Glioma samples retrieved from the CGGA database and the TCGA database. We clustered the gliomas into different groups by using expression values of Gβ and Gγ genes extracted from RNA sequencing data. The Kaplan–Meier method with a two-sided log-rank test was adopted to compare the OS of the patients between GNB2 group and non-GNB2 group. Univariate Cox regression analysis was referred to in order to investigate the prognostic role of each Gβ and Gγ genes. KEGG and ssGSEA analysis were applied to identify highly activated pathways. The “estimate” package, “GSVA” package, and the online analytical tools CIBERSORTx were employed to evaluate immune cell infiltration in glioma samples.Three subgroups were identified. Each subgroup had its own specific pathway activation pattern and other biological characteristics. High M2 cell infiltration was observed in the GNB2 subgroup. Different subgroups displayed different sensitivities to chemotherapeutics. GNB2 subgroup predicted poor survival in patients with gliomas, especially in patients with LGG with mutation IDH and non-codeleted 1p19q.The subgroup classification we proposed has great application value. It can be used to select chemotherapeutic drugs and the prognosis of patients with target gliomas. The unique relationships between subgroups and tumor-related pathways are worthy of further investigation to identify therapeutic Gβγ heterodimer targets. Glioma is a classical type of primary brain tumors that is most common seen in adults, and its high heterogeneity used to be a primitive feature for subgroup classification . Histori+ Channels, Phosphoinositide 3 Kinases, Mitogen-Activated Protein Kinases, and is also involved in microtubule polymerization, recycling endosomes, and Golgi fragmentation  and 12 γ-subunits  in the human body. βγ pairs are specifically related to downstream signals . Gβ1γ2 hhttp://www.cgga.org.cn) and 672 glioma samples retrieved from TCGA database (http://cancergenome.nih.gov/) were utilized in this study for reference. Relevant data included relapse samples. For the same patient, we only used the first tumor RNA sequencing data. The FPKM-standardized mRNA sequencing data was log2 transformed for all analyses. The count format mRNA sequencing data retrieved from TCGA was standardized by voom function.Nine hundred fifty-one glioma samples retrieved from the CGGA database (https://www.r-project.org/). PCA was employed to study the gene expression patterns in different glioma groups. We applied the first three PC values representing RNA sequencing data of each sample to establish a distribution map of the sample. Drug sensitivity analysis was later on performed with “pRRophetic” package (https://www.kegg.jp/kegg/pathway.html). The “estimate” package, “GSVA” package, and the online analytical tools CIBERSORTx (https://cibersortx.stanford.edu/) were employed to evaluate immune cell infiltration in glioma samples  and CGGA (98.6%) datasets .Publicly available datasets were analyzed in this study. This data can be found here: .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.


The inverseparticipation ratio is used to quantify the localization of phononmodes associated with the transition. We infer that suppression ofoctahedral rotations is an important factor to enhance defect tolerance.Improvement in theoptoelectronic performance of halide perovskitesemiconductors requires the identification and suppression of nonradiativecarrier trapping processes. The iodine interstitial has been establishedas a deep level defect and implicated as an active recombination center.We analyze the quantum mechanics of carrier trapping. Fast and irreversibleelectron capture by the neutral iodine interstitial is found. Theeffective Huang–Rhys factor exceeds 300, indicative of thestrong electron–phonon coupling that is possible in soft semiconductors.The accepting phonon mode has a frequency of 53 cm This behavior of halide perovskites has beenbroadly termed “defect tolerance”.9 Further improvementin the performance of halide perovskite devices requires suppressionof nonradiative carrier capture and recombination events.10 In this report, we perform a quantum mechanicalcarrier-capture analysis of the interaction between electrons andthe iodine interstitial in CH3NH3PbI3 (MAPI).The unusual defectchemistry and physics of lead halide perovskiteshas attracted significant attention.n or p type) semiconductor; minority carrier capture becomes the rate-limitingprocess. A necessary step is the change from delocalized-to-localizedelectronic wave functions, and multiple phonon (ℏω) emissionthrough the associated structural relaxation,11 as illustrated in 0), theoverall recombination process isShockley–Read–Hall (SRH) recombinationis associatedwith the successive capture of an electron and hole following thephotoexcitation  ensures conservation of energy. Each of these quantitiescan be derived from density functional theory (DFT) calculations.13Making use of Fermi’s golden rule, the carrier capture coefficientfrom an initial state ΔE and ΔQ refer to vertical and horizontal offsets in a configurationcoordinate diagram, and that ωi (ωf) is the frequency of the effective one-dimensional vibration inthe initial  charge state  as implemented in the GPU port of mt:17 whichhas been shown to accurately describe the structures and phonons ofthese materials. The internal atomic coordinates were relaxed untilthe force acting on each atom was less than 0.01 eV Å–1. Defect formation energies were converged to within 0.01 eV performula unit between the 192- and 384-atom supercells.So that no preference was given to a particularcombination ofoctahedral tilts the starting point for atomic relaxation was MAPIin the pseudocubic phase. The interstitial was placed in a 192-atomsupercell built from an expansion of the 12-atom unit cell, usingthe transformation matrix 18 (α = 0.43) includingspin–orbit coupling (SOC), with total energy converged to within10–5 eV. The electron–phonon coupling termis derived from wave functions calculated with the PBEsol functionaland SOC.The potentialenergy surface was calculated using the screened-exchangeHSE06 functional–8 eV,and the PBEsol functional. A 2 × 2 × 2 gamma centered Monkhorst–Packmesh was used for the Brillouin zone integration. To extract the phononeigenvectors and frequencies the Phonopy package19 was used. The Inverse Participation Ratio wascalculated using the Julia-Phonons package.20The harmonic phonon modes were calculatedusing a 2 × 2 × 2 supercell expansion . To evaluate the force-constant matrix thefinite displacement method was used with displacements of 0.01 Å.Forces were computed in VASP using a plane wave basis set of 700 eV,a total energy convergence criterion of 101 Interstitial iodine may be formed by the incorporationof excess iodine (I(g) ⇌ Ii) or through Frenkelpair formation (II ⇌ VI + Ii). There are three accessible charge states for the iodine interstitial(+/0/−). The calculated geometries of the three charge statesare shown in Common vacancydefects do not introduce levels into the band gapof lead iodide perovskites, while iodine interstitials do.2– complex. This is referredto as an H-center by the metal halide community and has a characteristicparamagnetic S = (1/2) spin configuration.21 Iodine is well-known to form polyiodide chainswith bond lengths that are sensitive to the charge state. The I–Ibond length in solid orthorhombic crystalline iodine is 2.67 Å,which lengthens to 3.23 Å upon formation of I2–.22 Our bond lengths are within 0.05 Å of this value. Owing tothe octahedral tilting pattern, we distinguish between in-plane (IP)and out-of-plane (OP) configurations for this defect.The neutral interstitial,rather than remaining as an isolatediodine atom in an interstitial region, bonds with a lattice iodineto form an I3–. For example, the tri-iodide groupin CsI3 has interbond distances of 2.82 and 3.10 Å,with the longer bond possessing the majority of the additional charge.23 In the negative charge state, the antibondingorbitals are filled, resulting in a split-interstitial configurationwith the longest I–I bond length of all three charge states.For thepositive charge state, an asymmetric trimer structure isfound that is typical of IF close to the valenceband) and the negative charge is favored in an n-type regime (EF close to the conduction band). The neutralinterstitial is metastable but has been suggested to form under illumination,25 which is our focus here.In the dark, the positive charge state is thermodynamically favoredin a p-type regime  hybrid density functional18 and incorporatingspin–orbit coupling (SOC), demonstrating the sensitivity ofthis system to the exact calculation parameters and defect geometries.As an extension to static DFT, ab initio molecular dynamics (MD) canbe used to model the motion at room temperature. Large fluctuationsin the halide vacancy defect energy level (up to 1 eV),26 and the formation of hole polarons that suppresscharge recombination27 have both been reported.However, a comprehensive MD analysis—using a hybrid functionaland SOC—remains computationally prohibitive.28 We find a charge capture rate that is highly sensitive to the defectgeometry; the importance of geometry relaxation, comparison with othercomputational reports, and the reference configuration for defectenergetics are discussed in the Supporting Information.Various first-principles studieshave attempted to elucidate thenature of charge capture at the iodine interstitial site. Fast nonradiativerecombinationQ is defined as i with mass mi and a displacement from equilibrium of Δri. After electron capture at IiOP, not only theI dimer but also the Pb atoms and the surrounding I atoms that formthe octahedra relax significantly, as shown in Figure S6). As the large displacements of the heavy Pb andI atoms are involved, ΔQ is large (ΔQ = 36 amu1/2 Å). This value is doublethat typically found for nonradiative recombination centers in kesterites15 and demonstrates that there is strong couplingbetween the electronic charge state of the defect and the latticedistortion.We now consider the potential energy surface (PES) associatedwithsequential electron and hole capture, Q isnot unique. For example, we might define ΔQ in the MAPI:Ii system as the root meansquared displacement of the two bonding iodine. For this definitionof ΔQ we find that electron trapping at theneutral iodine interstitial proceeds with a small geometrical rearrangement, ΔQ = 0.073 Å, resulting in fast radiative electroncapture. This small distortion of the iodine dimer has been reportedpreviously.24 However, this model excludesthe large relaxation of the surrounding perovskite structure, leadingto a significant underestimation of ΔQ and S.The definition of configuration coordinate Wif. Accepting modes take up theexcess electronic energy after charge capture, resulting in a changeof mean displacement. Rather than consider each phonon mode in turn, we use the effectivemode Q to consider the accepting modes only.29There are two classes of phonon mode associatedwith nonradiativetransitions. Promoting modes couple the initial and final electronicstates by producing a sizable electron–phonon coupling matrixelement Wif, a prefactorin Q, and (ii) the change in energy between these two states.For electron capture at the neutral iodine interstitial, Wif is 0.0036 eVamu–1/2 Å–1. This is comparable to an estimate for the maximum possible value, Wifmax = 0.0048 eVamu–1/2 Å–1,demonstrating that Q has both accepting and promotingcharacter, and justifying the use of the configuration coordinate.Further details of this comparison can be found in the Supporting Information.S in the strong coupling regime(S ≫ 1) is typically associated with nonradiativecarrier capture accompanied by multiphonon emission.29 The harmonic PES of the iodine interstitial is soft, withan effective frequency of 38 cm–1 (4.7 meV) and53 cm–1 (6.6 meV) for the neutral and negative states,respectively. The low frequency of the negative charge state, andlarge ΔE, gives a “giant” Huang–Rhysfactor of 350. For comparison, a substitutional Si atom in GaAs , the archetypal defect exhibitinglarge lattice relaxation, has a Huang–Rhys factor of S = 75 with ω = 81 cm–1 and ΔQ = 9 amu1/2 Å.30The Huang–Rhysfactor, ei) at the gamma point of the Brillouin zone,we calculate the inverse participation ratio (IPR):N is the number ofphonon modes.31 A fully localized phononmode has an IPR of 1. For a 96-atom supercell, a fully delocalizedmode has an IPR of –1, equal to the frequency of the negative charge state PES in theharmonic approximation. The agreement indicates that this resonantmode has a strong accepting carrier and will be active in the uptakeof excess electronic energy after the charge transition. Additionalanalysis of the phonon modes associated with the defective crystalcan be found in the Supporting Information.To identify the normal modes that are associated with latticerelaxation,we analyze the phonon dispersion of the negatively charged iodineinterstitial. For each phonon eigenvector .32 Unfortunatelythis model, while computationally economic and ideal for high-throughputtype studies, is not valid for the iodine interstitial due to theanharmonicity of the PES.It is evident from –17 cm3 s–1. Analysis of the vibrational wave functionsfor each PES shows that quantum tunnelling between the initial andfinal charge states is significant at energies below the classicalbarrier . Despite tunnelling,the capture coefficient is low due to the large electron capture barrier(600 meV). Fast radiative recombination, which is of the order 1 ×10–10 cm3 s–1 in MAPI,33 dominates in the harmonic approximation.In the harmonic picture, the iodineinterstitial has an electroncapture coefficient of 8 × 10–10 cm3 s–1, suggesting that nonradiative electron capture will compete withradiative capture. The electron capture barrier is 148 meV, much smallerthan in the harmonic case, yielding a larger capture coefficient.As in the harmonic picture, quantum tunnelling is significant . Measuring the electron capture rateat the iodine interstitial is difficult, as the trap density is hardto quantify and varies considerably with the material processing protocol.Nevertheless, a tentative approximation of the capture rate from arate constant of 2 × 107 s–134 and assuming a trap density of 1 × 1016 cm–335 yieldsa capture coefficient of 2 × 10–9 cm3 s–1. This estimate of the capture coefficientis within 1 order of magnitude of our calculated value for the anharmonicPES.In the more accurate anharmonic picture the electron capture coefficientis 1 × 10We observe a striking asymmetry between the electron capturebarrier(148 meV) and the hole capture barrier (924 meV). This is due to thelarge lattice relaxation associated with iodine rearrangement uponelectron capture. The asymmetry provides an explanation for the lowtrap-assisted recombination rate. Although electron capture at theneutral interstitial is fast, the subsequent capture of a hole atthe negative iodine interstitial is energetically inaccessible forthermal electrons.In conclusion, the softnature of halide perovskites results instrong electron–phonon coupling and a large displacement ofthe surrounding inorganic octahedra following electron capture. Thisrelaxation process leads to a giant Huang–Rhys factor and facilitatesfast nonradiative electron capture.2)2PbI3, FAPI36) and, more generally, in mechanically soft semiconductorsthat are prone to structural disorder . Defect tolerant perovskites  may be engineered by suppressing octahedralrotations in response to the changes in defect charge state. Thismight be achieved through elemental substitutions that result in localstrain fields37 or through defect engineering.38We expect to find similarlylarge, anharmonic lattice relaxationsin other perovskites where dynamic octahedral tilting is evident (e.g.,HC(NHOn the basis of our results, the electroncapture process at theneutral iodine interstitial in MAPI is irreversible; it is energeticallyunfavorable for the electron to be released into the band or annihilatedby a hole. An extension of this procedure to cover all native defects,while being computationally demanding, would help to understand thenature of nonradiative losses in halide perovskites solar cells, aswell as avenues to further enhance efficiency toward the radiativelimit.


All patients who underwent an elective loop ileostomy formation were included.Distance from the ileocecal valve and surgical technique was applied as per surgeons’ preference, and loperamide management was not standardised. All FU data were recorded during in-person clinic visits. Photographs were taken of patients’ loop ileostomy after their written consent at standardised time points during the study, to reassure an objective assessment over the two centres.P ≤ 0.05 was considered as the significance level.Means ± standard deviation or median with interquartile range was used according to distribution, T-test, or Mann–Whitney U test to compare numerical data and Chi-square or Fisher’s exact for categorical data. Mean hospital stay of the 81 included patients was 11 ± 9 days. None of the patients had a history of/or concomitant small bowel resection.p = 0.03). There was a delay in discharge due to stoma-related issues in 17 patients (21%); because of HOS in 9 and stoma care in 8 patients (stoma training/application problems).No ileostomy-related reinterventions were recorded during admission. Mean stoma output at discharge was 696 ± 412 ml/day, with 27/81 (33%) being on loperamide  had an early ileostomy closure because of related morbidity .There were 12 readmissions in 8 patients (10%) because of ileostomy-related complications, with in 4/8 patients (50%) because of ileostomy output-related complications and need for intensive care unit admission in one patient within a week after discharge (stoma output at discharge 975 ml/day)  and need for intensive care unit admission in one of them within 1 week after discharge. Additionally, 9 patients (11%) experienced a delay in discharge during elective admission because of HOS which is a known risk factor for readmission.4HOS-related complications together with postoperative ileus/obstruction and deep infections are seen as the main reasons for readmission after elective colorectal surgery.With those observations in mind, our centres put forward a flowchart to aim for consistent stoma output management during admission and discharge with a focus on loperamide therapy Fig. . In our 5The importance of careful management of patients’ ileostomy output is further supported by a possible impact on their long-term kidney function.Strength of our study is the prospective design which allowed a detailed assessment of the ileostomy-related measures and a weakness the limited number of patients.Further research has to focus on the question if more consistent use of loperamide therapy during admission and FU can further reduce readmission rates and morbidity associated with ileostomy formation.


The influence of antipsychotic drugs on tumor necrosis factor-α (TNF-α) levels is unclear, and there is no consensus on the association between TNF-α and psychotic symptoms. This study aimed to investigate the differences in TNF-α levels and clinical correlations in first-episode drug-naïve (FEDN) patients with schizophrenia before and after treatment and in chronic patients.A total of 103 (51 FEDN and 52 chronic) patients and 114 healthy controls were recruited. Demographic and clinical data, including TNF-α levels, were recorded. We used the Positive and Negative Syndrome Scale (PANSS) to measure the psychopathology of all patients.TNF-α levels before treatment were significantly higher in FEDN patients than in chronic patients and healthy controls. No significant sex differences were found in the TNF-α levels of patients with schizophrenia. The TNF-α levels before treatment were significantly positively related to changes in PANSS negative symptoms in FEDN patients. The TNF-α levels in chronic patients were significantly negatively correlated with the general psychopathology subscales and PANSS total scores.Increased TNF-α levels in FEDN patients and their correlation with psychopathology indicate that inflammatory cytokines may play a crucial role in the etiopathogenesis of schizophrenia, and inflammation-directed therapy may, therefore, improve negative symptoms. Toxoplasma gondii, influenza, and cytomegalovirus) is associated with schizophrenia in adult children [Schizophrenia is a complicated and serious mental illness that mostly develops in early adulthood or late adolescence. The point prevalence of global age-standardized for schizophrenia was estimated to be 0.28% in 2016, and global epidemic cases rose from 13.1 million in 1990 to 20.9 million cases in 2016. Globally, schizophrenia leads to the population experiencing 13.4 million years of life living with disability [children . Nonsterchildren . The trachildren . These rTNF-α participates in the process of neuro-immune regulation and autoimmunity, including the regulation of development, repair, signal feedback, differentiation of glial cells during early growth, and maintenance of normal brain morphology . TNF-α sAlthough there is discrepancy among several studies, some meta-analyses have shown that patients with first-episode psychosis have higher TNF-α levels than normal controls , 20. HowThere are no consistent results on the relationship between TNF-α and psychiatric symptoms in patients with schizophrenia or on the relationship between changes in the TNF-α levels and psychiatric symptoms. One study found there was no association between TNF-α and psychiatric symptoms at admission and discharge in patients with schizophrenia . HoweverTherefore, the aims of our research were (1) to compare differences in the TNF-α levels among first-episode drug-naïve (FEDN) patients with schizophrenia, chronic patients, and healthy controls; (2) to assess TNF-α changes in FEDN patients before and after treatment with risperidone for 12 weeks and analyze the correlation between the TNF-α levels and psychiatric symptoms in patients with schizophrenia; and (3) to analyze the possible existence of sex differences in the TNF-α levels in patients with schizophrenia.Subjects in the schizophrenia group were admitted to the Beijing HuiLongGuan Hospital from January 2015 to December 2018. The healthy subjects were enrolled from the local community in Beijing. After we posted the recruitment information online, all healthy controls that met the inclusion criteria were recruited without any selection. All recruited patients were inpatients. All patients recruited in the study met the diagnostic criteria for schizophrenia spectrum disorders of the Diagnostic and Statistical Manual of Mental Disorders . All subjects were of Han nationality and aged between 18 and 60 years.The inclusion criteria for FEDN patients with schizophrenia were as follows: (1) no prescription antipsychotic medication before enrollment; (2) total score ≥ 60 on the Positive and Negative Syndrome Scale (PANSS); and (3) total duration of illness ≤5 years at first onset. The inclusion criteria for chronic patients with schizophrenia were as follows: (1) total duration of illness ≥60 months and (2) steady dosage of oral antipsychotic medication ≥12 months before the start of this study.The exclusion criteria for patients included (1) severe central nervous system disease, physical illness, immune disease, and recent administration of any immunomodulators; (2) drug or alcohol dependence, illicit drug use such as marijuana in the past 30 days; and (3) pregnancy or lactation. In total, 103 patients were enrolled, including 77 male and 26 female patients, with an average age of 37.65 ± 12.20 years; the mean duration of illness was 154.47 ± 144.65 months, and the average years of education was 10.61 ± 3.14 years.The inclusion criteria for healthy controls were as follows: (1) 18–60 years old; (2) no medical history of mental diseases, including schizophrenia, bipolar disorder, or substance dependence; no family history of mental illness at present or in the past; and (3) good physical health status, without serious physical diseases, immune diseases, or organic brain diseases, and without recent administration of any immunomodulators. The exclusion criteria for healthy controls were the same as those for the patient group. A total of 114 subjects were recruited, including 21 male and 93 female patients, with a mean age of 44.31 ± 14.40 years; the average years of education was 8.49 ± 3.96 years.Trained research staff administered a detailed questionnaire to all subjects to collect their sociodemographic data, general information, and medical status. We obtained an entire medical history, physical examination, and laboratory tests from all the participants, excluding those subjects who had abnormal examinations or medical illnesses, as mentioned in the previous exclusion criteria. Our research was approved by the Ethics Committee of Beijing HuiLongGuan Hospital; all enrolled participants and/or their legal guardians signed the informed consent form.FEDN patients were administered an oral monotherapy of risperidone (4–6 mg) and were prohibited from using other antidepressants and mood stabilizers. Patients with extrapyramidal syndrome could be treated with diphenol hydrochloride. A PANSS assessment was performed to evaluate the psychopathological symptoms of chronic patients and FEDN patients with schizophrenia before and after 12 weeks of treatment with antipsychotics. The PANSS contains a total of 30 items, among which 7 items are related to positive symptoms, 7 items to negative symptoms, and 16 items to general psychopathology. Each item is scored on a scale of 1 (none) to 7 (extremely severe) for psychopathological symptoms. After assessment by two psychiatrists with prior PANSS training, the intra-group correlation coefficient was > 0.80.On the second day after enrollment, following an overnight fast (avoiding food for > 10 h), blood samples were collected at 6:00–8:00 AM from the forearm veins of the chronic patients and healthy controls, and from the FEDN patients with schizophrenia before and after 12 weeks of antipsychotic treatment. After storage at room temperature for 1 h, the collected serum was centrifuged for 10 min at 3000 r/min and stored in a refrigerator at − 80 °C for examination. TNF-α levels were measured using a commercially available enzyme-linked immunosorbent assay kit . The same researcher tested the serum samples for all tests. The sensitivity of detection was 4 pg/mL, and the coefficients of variation inter-assay and intra-assay were 9 and 6%, respectively.t-tests were performed to compare differences between the groups in terms of their consecutive demographic variables. We used the Kruskal-Wallis H test to analyze differences between the groups that had non-normally distributed variables. The correlation between the TNF-α levels and psychotic symptoms was analyzed using Pearson or Spearman correlations. Nonparametric tests of two related samples were used to compare the TNF-α levels before and after treatment. Multivariable linear regression analysis was used to analyze the correlations between the TNF-α levels and demographic or psychiatric symptoms. We used a generalized linear model to analyze the relationships between non-normally distributed variables and the clinical data. The generalized linear mixed model was used to test the effect of TNF-α on psychopathology before and after treatment. Statistical graphing was performed using GraphPad Prism 7.0 software. We applied the Bonferroni correction to adjust for multiple testing. All p-values were two-tailed, with a significance level of < 0.05.SPSS software version 22.0 was used to perform the statistical analyses. We applied the one-sample Kolmogorov-Smirnov test to examine the normality of data. The χ2 test was used to analyze the differences between the categorical variables, and one-way ANOVA or independent sample p < 0.001). The results of the post-hoc pairwise comparisons revealed significant differences in sex among the three groups. There were more males than females among the FEDN patients and chronic patients and more females than males among the healthy controls (p < 0.05). The age of FEDN patients was significantly lower than that of chronic patients and healthy controls (p < 0.001), whereas there was no significant difference in age between chronic patients and healthy controls (p > 0.05). The education level of FEDN patients was significantly higher than that of chronic patients and healthy controls (p < 0.05), but there was no significant difference in education level between chronic patients and healthy controls (p > 0.05). There was no significant difference in the age of onset between FEDN patients with schizophrenia and chronic patients (p > 0.05), and significant differences were observed in the duration of illness, PANSS total score, and the three subscale scores between the two groups (p < 0.05).As shown in Table p < 0.001; Table p < 0.05). Further pairwise comparison analyses showed that the TNF-α levels in FEDN patients before treatment were significantly higher than those in chronic patients and healthy controls (p < 0.05). The significant differences in FEDN patients and healthy controls passed the Bonferroni correction (p < 0.0167). No significant difference was found in the TNF-α levels between chronic patients and healthy controls (p > 0.05). In the pairwise comparisons of the generalized linear model, significant differences were noted in the TNF-α levels among the subjects of different ages, and the TNF-α levels in the lower age group were higher than those in the higher age group (p < 0.01). There was no interaction between age and group (p > 0.05). There was no significant difference in the TNF-α levels among subjects with different education levels (p > 0.05).First, the normality test showed that the TNF-α levels in FEDN patients before treatment and in normal controls did not conform to a normal distribution. The TNF-α levels in chronic patients were normally distributed. The Kruskal-Wallis H test revealed significant differences in the TNF-α levels between patients with schizophrenia and healthy controls p < 0.00; Table 1p > 0.05). The generalized linear model was conducted with the TNF-α level as the dependent variable and sex, age, and education levels as the predictor variables for FEDN patients and chronic patients. The result showed no significant differences in the TNF-α levels between males and females .To analyze possible sex differences in the TNF-α levels in patients with schizophrenia, we used a stratification analysis, which revealed no significant differences in the TNF-α levels between males and females in the three groups (p > 0.05). After controlling for sex, age, and education level, the regression analysis still showed no significant relationship between the TNF-α levels and psychotic symptoms in FEDN patients before and after treatment (p > 0.05). The partial correlation analysis showed that the TNF-α levels in chronic patients were significantly negatively correlated with the PANSS total scores  and PANSS general psychopathology subscale scores  after controlling for age, sex, and education (Table p < 0.05).The Spearman correlation analysis showed no significant associations between the TNF-α levels and psychotic symptoms  in FEDN patients before and after treatment . The Spearman correlation analysis showed that reductions in the TNF-α levels before and after treatment were significantly positively associated with PANSS negative symptoms before treatment  and positively correlated with PANSS negative subscale reductions . We used the generalized linear mixed model to test the effect of TNF-α on psychopathology before and after treatment, with age, sex, and education level as covariables. Our results showed that the TNF-α levels before treatment were significantly positively correlated with changes in PANSS negative symptoms .The mean TNF-α level in FEDN patients with schizophrenia was 24.95 7.48, 43.12) pg/mL after treatment. The nonparametric Wilcoxon test (for testing two related samples) was used to compare differences in the TNF-α levels of FEDN patients before and after 12 weeks of treatment with antipsychotic drugs, and there was no significant difference  This study was not a randomized controlled study; thus, there may have been bias in the selection of the sample, and scientific causal inferences cannot be made. In the future, longitudinal studies that are more scientific should be designed with larger sample sizes in order to explore the relationship between TNF-α and psychotic symptoms. (2) The potential effects of various antipsychotics on the TNF-α levels were not assessed in the study. Therefore, future studies should include patients who use different antipsychotics to compare the effects of different drugs on inflammatory factors in each group. (3) Many inflammatory cytokines are involved in the inflammatory process in humans. Different inflammatory factors have mutual effects, and the levels of inflammatory cytokines in different fluids, such as plasma and cerebrospinal fluid, are different. Therefore, in future research, the levels of various inflammatory factors in the cerebrospinal fluid or plasma should be evaluated to more comprehensively explore the potential role and mechanisms of inflammatory factors in the pathogenesis and efficacy of schizophrenia. (4) PANSS has some limitations in assessing the avolition-apathy domain, which is a dimension of negative symptoms. So the Brief Negative Symptom Scale (BNSS) and the Clinical Assessment Interview for Negative Symptoms (CAINS), as well as other scales, may be used to assess the primary and secondary negative symptoms in the future .Significantly elevated TNF-α levels in FEDN patients and the correlation between TNF-α levels and psychotic symptoms in patients suggest that inflammatory cytokines are involved in the formation and alteration of psychopathic symptoms, especially negative symptoms. At present, the pharmacological mechanism of schizophrenia mostly focuses on neurotransmitters such as dopamine, glutamate, and serotonin. Antipsychotic drugs also improve psychotic symptoms by alleviating the imbalance of these neurotransmitters. However, although the patients received a standardized medical treatment, the effect was still not satisfactory. Some patients had prominent negative symptoms, and their social functioning could not be maintained well. The role of immune functions and neuroinflammatory responses in the pathogenesis of schizophrenia is still worth further investigation, which may provide some targets for the exploitation of new antipsychotic drugs.