We used two novel methodologies: A cross-lagged panel model (CLPM) to test between-person changes and a trait-state-occasion model (TSO) to test within-person changes. We found that both the CLPM as well as the TSO models showed that gratitude at work prospectively predicted further lower workplace materialism. Specifically, the CLPM shows that individuals with higher than average gratitude at Ti, are more likely to show lower than average materialism at Ti+1. The TSO shows that individuals with a higher than their usual level of gratitude at Ti are more likely to show a lower than their usual level of materialism at Ti+1. Important implications for materialism research as well as for the Positive Organizational Psychology field are discussed.Materialism at work refers to a higher importance attached to extrinsic  versus intrinsic  employees’ ‘aspirations’. Research from self-determination theory has consistently found that materialism at work is strongly detrimental for both employees and organizations. For example, materialism is negatively associated with lower job satisfaction and engagement and positively associated with higher turnover intentions and job insecurity. Unfortunately, there are no viable strategies for reducing materialism in the workplace yet. In this sense, based on emergent research in psychology, we theorized that dispositional gratitude—a key construct within the Positive Organizational Psychology field—could be a protecting factor against materialism. Further, we conducted a three-wave longitudinal design among a large sample of Chilean workers ( Workplace materialism refers to a higher importance attached to extrinsic  versus intrinsic  aspirations at work ,2. Self Unfortunately, there are no viable strategies for reducing materialism in the workplace yet. Researchers have focused on the negative consequences of materialism instead of its antecedents . Howevern = 1841). Using novel methodologies, we tested a cross-lagged panel model (CLPM) as well as a trait-state-occasion model (TSO). Whereas a CLPM test between person differences, a TSO test within-person differences. Theoretical and practical implications might emerge from our research, especially for materialism research as well as for the Positive Organizational Psychology (POS) field.To test our hypothesis, we conducted a three-waves longitudinal design, among a large sample of Chilean workers 021] focuThe Kasser and Ryan ,23 approResearch has consistently shown that higher materialism is strongly associated with lower well-being  as well as with higher ill-being . Thesome employees view their jobs as opportunities to exercise their competencies and skills, pursue personal interests and make meaningful contributions to society” (intrinsic aspirations) other employees may “focus primarily on financial success, having control and influence over others and occupying a prestigious position at work” (extrinsic aspirations) ; 42.4% had a salary between $1414.4 and $2828.3 ; 14.4% had a salary between $2828.3 and $4242.4 ; 4.2% had a salary between $4242.4 and $5373.8 ; and 2.6% had a salary higher than $5373.8 .χ2 (2)] = 0.72, p = 0.070]), gratitude at work ([t(1839)] = −1.66, p = 0.097]) and materialism at work ([t(1839)] = 0.34, p = 0.073]) between those respondents whose left the survey after responding Wave 1 versus the rest of participants. We only found significant differences in terms of age ] = −4.59, p < 0.001]). Little MCAR test [χ2 (180)] = 205.030, p = 0.097]). Thus, we included all of the 1841 participants in our analyses, using full information maximum likelihood (FIML), to handle missing data [Regarding attrition , no statCAR test  showed ting data .Regarding normality, several authors agree that despite there is no clear rule for cut-off criteria, a conservative guideline assumes that multivariate normality should not be a problem if the threshold of the absolute values of skewness and kurtosis does not exceed 2 and 7 respectively ,64,65. Tn = 1841) fulfills previous requirements.Regarding our sample size, we considered it is enough to test our SEM models appropriately. Indeed, it exceeds the ranges suggested for previous research. For example, Wolf, Harrington, Clarks and Miller  have claWe used constructs with good psychometric properties. The original scales were translated using a standard back translation procedure .We used a short version of the Aspiration Index  adapted We adapted the 6 items dispositional gratitude scale (GHQ-6) developed by McCullough et al.  to the wConsidering the benchmarks raised by Hu and Bentler , Kline  and Tabaχ2 (54) = 2878.32, p < 0.001) is significantly worse than a model where gratitude (6 indicators) and materialism (6 indicators) were modeled as two different latent variables (χ2 (53) = 1734.58, p < 0.001), Δχ2 (1) = 1143.74, p < 0.001. At T2, the collapsed model (χ2 (54) = 1556.93, p < 0.001) is significantly worse than the two-factor model (χ2 (53) = 1059.01, p < 0.001), Δχ2 (1) = 497.92, p < 0.001. At T3, the collapsed model (χ2 (54) = 1073.39, p < 0.001) is significantly worse than the two-factor model (χ2 (53) = 808.711, p < 0.001), Δχ2 (1) = 264.68, p < 0.001.We started with a confirmatory factor analysis (CFA) in order to examine the factorial validity of our core measures  in each assessment time. At T1, results showed that the collapsed model , and the model showed a good fit to the data, χ2 (282) = 1,356,155, p < 0.001, RMSEA = 0.045 , CFI = 0.91, SRMR = 0.07.First, we set up a six-factor measurement model for gratitude and materialism. As suggested by Joreskog , we incoThe results of our CFAs analyses and the measurement model give us confidence in terms of the validity of our constructs in all waves. Thus, we proceeded to test the CLPM in Model 1 and the TSO in Model 2.Model 1: Cross-lagged panel modelχ2 (286) = 138,788, p < 0.001, RMSEA = 0.046 , CFI = 0.91, SRMR = 0.071. Second, to test invariance, we constrained all factor loading of each latent variable to be equal across waves. Model fit was also acceptable χ2 (300) = 140,334, p < 0.001, RMSEA = 0.045 , CFI = 0.91, SRMR = 0.073. Because the reduction in CFI is less than 0.01, the assumption of invariance was tenable  was regressed on its own lagged measure at (T) as well as on the other lagged measure at (T) ,76. Thus= 0.001; ), and thχ2 (304) = 141,082, p < 0.001, CFI = 0.91, SRMR = 0.074, RMSEA = 0.044 . Additionally, this model showed no significant differences compared with the previous model, Δ χ2 (4) = 7.47, p = 0.11. Factor loadings ranged from 0.33 to 0.89 . Supporting H1, gratitude at work was a significant negative predictor of materialism at work at the between-person level, β = −0.07, , p = 0.001. However, H3 was not supported. Materialism at work did not predict gratitude at work, β = −0.03, , p = 0.16. No other significant paths were found . The structural parameters are presented in Third, and finally, aiming for a more parsimonious model as well as to gain statistical power, we constrained all paths to be invariant over time . This fiModel 2: Expanded Multivariate Latent Trait-State-Occasion ModelTo complement the results obtained in CLMP model, we conducted a TSO, following Unanue, Martela, Vignoles, and Dittmar . This meχ2 (297) = 1371.18, p < 0.001, CFI = 0.91 SRMR = 0.071, RMSEA = 0.044 . In second instance in order to increase the statistical power, we constrained all autoregressive and cross-lagged paths to be invariant over time [χ2 (301) = 1381.10, p < 0.001, CFI = 0.91, SRMR = 0.073. RMSEA = 0.044  however, it differed significantly from the constrain model, (Δ.χ2 (4) = 9.92, p = 0.04). Inspection of all the respective paths found that the stability path of materialism needs to be estimated freely. This final model showed an adequate fit, χ2 (299) = 1374.45, p < 0.001, CFI = 0.91, SRMR = 0.073. RMSEA = 0.044  and did not differed significantly from the original model, Δ χ2 (2) = 3.27, p = 0.19. Factor loadings ranged from 0.38 a 0.92 .We started with a 6-factor model with constrained loadings. This constrained model showed an acceptable fit, ver time . This moβ = −0.20, , p < 0.01. Thus, H2 was supported. We did not find support for H4. Materialism at work was not a prospective predictor of gratitude at work, β = −0.04, . Details may be seen in Based on this final model, we found that gratitude at work prospectively predicts lower materialism at work at the within-person level, SDT  has consFollowing our first and second hypotheses, prospective analyses were conducted considering the prospective relationship between gratitude and materialism based on between-person analysis (CLPM) and a within-person analysis (TSO). Concretely, these two types of analysis were carried out because both interpret the longitudinal relationships between the variables in different ways. For example, previous studies that used CLPM and TSO models have observed that sometimes these relationships can vary or even show opposite results depending on the type of analysis performed ,82. HoweIn summary, the negative relationships observed in both types of longitudinal analysis between gratitude and materialism suggest that interventions and programs oriented to increase gratitude in work contexts could promote a decrease in this type of aspirations. According to the previous literature, gratitude is an emotion that from an evolutional perspective, contributes to better interpersonal relationships that are stable over time because gratitude is strongly related to prosociality ,84. Thisa priori results may seem surprising as materialism, in addition to being negatively related to different work well-being, is also often associated with lower-quality relationships [Hypotheses 3 and 4 aimed to determine whether materialism can also explain prospectively and negatively gratitude in workers. This aspect should be considered as aspirations are long-term objectives that directly influence our choices and lifestyles . In thisionships ,89. Mateionships ,90. HoweThe results show that gratitude can contribute to a decrease in the materialism of workers across time but delving into the variables may promote that these workers experience more gratitude at their positions. As indicated by previous studies, gratitude at work can be increased through programs and interventions, but it is necessary to understand if that trait can be developed and then maintained over time. Thus, more longitudinal studies are required on this specific topic.First, it remains to be demonstrated whether the impact of gratitude on materialism have the same effects in an unexplored and specific domain: The workplace. To date, research has been conducted only in general life settings or in specific domains such as schools . Thus, wWe hope our results may help academics and practitioners in the Positive Organization Psychology field (POS) to find new ways of improving workers’ quality of life, as well as building healthier and more resilient organizations . Indeed,Positive psychology as well as the POS field has developed outstanding strategies to increase gratitude in organizations. In the present section, we will outline three of them. First, following Seligman et al.  we recomSecond, following Emmons and McCullough  organizacomposed of episodic gratitude at the event level, persistent gratitude at the individual level, and collective gratitude at the organizational level” in order to build a “grateful workplace” (p. 361). The authors strongly recommend that organizations develop formal and measurable gratitude programs. Among some practices, Fehr and colleagues suggested developing appreciation programs, beneficiary contact exercises, developmental feed-back programs, human resources’ alignment, and closer contact between employees and customers (among others), which may foster the three kinds of gratitude already mentioned, but also increases workers well-being and attitudes, as well as a company’s performance.Third, and finally, Fehr et al.  developeStrategies aimed to decrease materialism should focus on (a) decreasing the importance attached to extrinsic goals, (b) increasing the importance attached to intrinsic goals, or a combination of (a) and (b). These strategies are not easy at all, because we live in a consumer culture  that shoScience has shown that extrinsic and intrinsic goals are a kind of “see-saw” . For exaResearch has also shown that materialism increases when people feel insecure and have worries about their self-worth ,97. WhenPrevious recommendations point toward a change in employee’s life style. However, leaders and companies also have a big responsibility in decreasing materialism at work. Indeed, it has been argued that materialism is present in the value system of several organizations, due to strongly encouraging the pursuit of extrinsic goals such as productivity, efficiency and profitability despite the costs it has on worker´s quality of life ,99. UnfoFirst, leaders and organizations should develop formal policies to encourage the pursuit of intrinsic values . Second, it has been shown that exposing workers to materialistic messages could increase the pursuit of extrinsic values, which in turn may increase workplace materialism ,101. FurLike all kinds of research, we recognize that we also have some limitations. First, the inclusion of non-self-reported measures is an important element that should be considered in future studies. However, to date, the most stablished method to capture gratitude and materialism is through self-reporting. ,38. AddiSecond, although our study helps to estimate the prospective relationships between gratitude and materialism, it does not exclude the option of third variables excluded  that may be playing a role. Third, despite the fact that prospective/longitudinal associations are a key requirement for establishing causality, this kind of design cannot test causal associations properly. Thus, we encourage further research to conduct experimental designs. Fourth, our sample does not allow cross-cultural generalizations. Future research should explore different context and cultures. Nonetheless, our research in Chile advanced significantly the existing knowledge regarding the studied variables. Fifth, we collected demographic data only for age, gender, and salary. That is because we were interested in studying working adults in general, without any restrictions. Nonetheless, we recognize that it would be important to have more information about a participant´s profile such as the kind of company they work for ; whether or not participants work for public or private organizations; whether or not they have managerial responsibilities and the specific job area of the participants . Sixth, and finally, it would be important to reduce attrition rates in future research. However, this issue is common when using longitudinal designs ,103. ForThe results show negative prospective relationships between gratitude at work and workplace materialism considering two levels of analysis, namely between- and within-person. This implies that people with high gratitude at work exhibit less materialism across time. In turn, people who score high on gratitude at work also show lower levels of materialism than their peers. These results reinforce the relevance of programs and interventions aimed to foster gratitude at work in order to reduce the materialistic aspirations of workers. On many occasions, organizations promote reward systems that focus on success and prestige at work. However, according to the literature, this has been related to lower levels of work satisfaction and a higher perception of work overload. Concretely, materialistic people tend to be more focused on strengthening their fame and image, and consequently their own self, whereas gratitude promotes better interpersonal relationships and is an emotion centered on collective well-being. In this sense, the results of this study show that gratitude can reduce the materialism of workers across time. Nevertheless, further studies are necessary to delve into the prospective relationship between the gratitude of workers and other variables associated both to organizational climate and the welfare of workers.


To characterise peritumoral zones in glioblastoma and anaplastic astrocytomaevaluating T2 values using T2 mapping sequences.In this study, 41 patients with histopathologically confirmed World HealthOrganization high grade gliomas and preoperative magnetic resonance imagingexaminations were retrospectively identified and enrolled. High gradegliomas were differentiated: (a) by grade, glioblastoma versus anaplasticastrocytoma; and (b) by isocitrate dehydrogenase mutational state, mutatedversus wildtype. T2 map relaxation times were assessed from the tumourcentre to peritumoral zones by means of a region of interest and calculatedpixelwise by using a fit model.P = 0.0049 –fit-model: glioblastoma –25.02± 19.89 (–54–10); anaplastic astrocytoma–5.57±22.94 (–51–47)). Similar results were found when the cohort wassubdivided by their isocitrate dehydrogenase profile, showing an increaseddrawdown from tumour centre to periphery for wildtype in comparison tomutated isocitrate dehydrogenase (P = 0.0430 – fit model:isocitrate dehydrogenase wildtype –10.35±16.20 (–51) – 0; isocitratedehydrogenase mutated 12.14±21.24 (–15–47)). A strong statistical proof forboth subgroup analyses  was found.Significant differences between T2 values evaluated from the tumour centre tothe peritumoral zone were found between glioblastoma and anaplasticastrocytoma, showing a higher decrease in signal intensity  fromtumour centre to periphery for glioblastoma (Peritumoral T2 mapping relaxation time tissue behaviour of glioblastomadiffers from anaplastic astrocytoma. Significant differences in T2 values,using T2 mapping relaxation time, were found between glioblastoma andanaplastic astrocytoma, capturing the tumour centre to the peritumoral zone.A similar curve progression from tumour centre to peritumoral zone was foundfor isocitrate dehydrogenase wildtype high grade gliomas in comparison toisocitrate dehydrogenase mutated high grade gliomas. This finding is inaccordance with the biologically more aggressive behaviour of isocitratedehydrogenase wildtype in comparison to isocitrate dehydrogenase mutatedhigh grade gliomas. These results emphasize the potential of mappingtechniques to reflect the tissue composition of high grade gliomas. Furthermore, IDH mutation helps to distinguish between primaryand secondary GBMs, as the mutation is rarely seen in de novo GBMs.Glioblastomas (GBMs) and anaplastic astrocytomas (AA3s) represent the majority ofhigh grade gliomas (HGGs) and are the most common malignant adult brain tumours. Fortreatment, prediction and prognostic decision HGGs have to be classified. The goldstandard therefore is the World Health Organization (WHO) classification. Withinthis classification, HGGs are categorised according to the histological morphologyand since the last update in 2016 to genotypic parameters as they also have animpact on predicting the outcome. AA3s are categorised as WHO grade III and GBMs asWHO grade IV tumours.3As a reaction to these findings, the WHO classification included the genotypicparameters in the revised version from 2016.5Within GBMs, another well-known molecular parameter with a reported predictive valueis the methylation of the methylguanine methyltransferase (MGMT) promotor gene.Patients with a methylated MGMT promotor benefit from adjuvantchemotherapy. However, the detection of any relapse situation after resection remainsdifficult. The reason for this might be that the majority of recurring HGGs iswithin 2–3 cm of the resection margin. This aspect reflects the importance of theperitumoral zone, which is the active spot of tumour growth and response. With this the preoperative visualisation of the infiltrative peritumoral zoneis crucial for planning the resection margin and subsequently directly affecting thefrequency of margin recurrences. The major problem with this zone is that the peritumoral zone may representfluid-filled compartments due to the disruption of the blood–brain barrier (BBB)and/or accumulation of active tumour cells. According to Lemee et al. the peritumoral zone is radiologically defined asthe healthy, magnetic resonance non-enhancing brain area a few centimetres aroundthe tumour. It is usually T2-hyperintense, representing vasogenic oedema, which mayindicate an infiltration of tumour cells. To date it is not possible to distinguish between peritumoral oedema orinfiltration of tumour cells by using the ‘established’ magnetic resonance imaging(MRI) features. No ‘newer’ MRI technique (diffusion weighted imaging (DWI) andapparent diffusion coefficient (ADC), susceptibility weighted imaging (SWI), dynamiccontrast enhancement (DCE), spectroscopy, etc.) enables a differentiationsufficiently. Furthermore, this zone is of great interest, regarding the differentaggressive behaviour in tumours within the same WHO grade. It has been describedelsewhere that isocitrate dehydrogenase wildtype (IDHw) AA3 HGGs show a moreaggressive behaviour than isocitrate dehydrogenase mutated (IDHm) AA3 HGGs. This might originate from more active tumour cells and with thistheoretically directly influencing the resection margin. If IDHw are treatedsimilarly to IDHm AA3 in accordance with their WHO grade, early recurrence mightincrease.The management of such highly aggressive tumours as GBMs is difficult. Thedevelopment of reliable and easy to realise response criteria is challenging. Todate, the gold standard for response assessment is the response assessment inneuro-oncology (RANO) criteria.1114 This might have the potentialto distinguish between tumour cells and vasogenic oedema in the peritumoral zone.Recent studies report an association between T2 mapping relaxation times and themolecular background in gliomas.Advanced MRI mapping techniques quantify the relaxation time at different echo timespixelwise and are able to give more accurate information about tissue structure andcomposition. Usually the magnetic resonance image contrast is determined by thespecific T2 relaxation times. Oedema, tumour infiltration and an increased bloodflow or water diffusion processes are known to influence the image contrast inT2-weighted images. A quantitative approach measuring the T2 relaxation times asabsolute numbers in milliseconds (ms) and coding them colour or gray-scaled, alsoknown as T2 maps, seems to be the most reliable, objective and reproducible imagingtechnique to visualise the image contrast in T2-weighted/fluid-attended inversionrecovery (FLAIR) images.The purpose of this study was to characterise and gain more insights into theperitumoral zones of GBMs and AA3s according to their IDH profiles. Therefore, T2values have been evaluated, at different echo times using T2 mapping sequences.n = 22) and a AA3(n = 19) cohort. Histopathological reports were availablefor all patients and IDH status was determined by immunohistochemistry. Thestudy protocol  conforms to the ethicalguidelines of the 1975 Declaration of Helsinki. Some of the patients included inthe present analysis participated in a previous study.A total of 41 patients with either singular GBM or AA3 were enrolled from April2015 to December 2016. Patients younger than 18 years or with prior surgery wereexcluded. Patients were divided into a GBM  or on a3T scanner . MRI of the brain consisted of anaxial T1-weighted sequence, repetition time (TR) 550 ms, echo time (TE) 8.9 ms,slice thickness 5 mm; in-plane resolution 0.8984 mm × 0.8984 mm, acquisitionmatrix 256 × 216), T2 fat saturation (T2-fs) axial  186 × 230 rows, in-plane resolution0.4492 mm × 0.4492 mm), axial FLAIR sequence , T2 mapping  and magnetisation-prepared rapid gradient echo (MPRAGE)sequence post contrast . T2 maps were reconstructed by using a voxelwise, monoexponentialnon-negative least-squares fit analysis  witha voxel size of 1.9 × 1.0 × 3 mm³.Images were postprocessed with the visage software tool . T2 map ROIs were drawn manually by an experiencedneuroradiologist (>10 years of experience) plus a second reader (2 years ofexperience) with a standardised diameter of 5 mm in one representable slice. TheROIs were placed radiating out from the centre of the tumour in one line outinto the peritumoral zone/oedema with a maximum of three ROIs around the tumour.The ROIs covered representable tissue of a peritumoral zone, while areas ofnecrosis and vessels were excluded . The slice with the largest diameterof peritumoral T2-weighted/FLAIR hyperintensity was chosen, the anatomicalcentre of the tumour was delineated. Another ROI was placed in thehealthy-appearing white matter of the contralateral lobe using an imageprocessing program to ensure reliability of the measurements .R2) withan intercept range between 0 and 1 was calculated. The stretching factor of thesecond-degree fit is a measure of the increase of T2 across the tumour and wasevaluated in every subject and presented in the model function. The coefficientsof determination and stretching factors for both groups were compared using theMann–Whitney U-test.The data were analysed by using XLSTAT, version 2011.3.01 . The Mann–Whitney U-test was used as a two-tailed test to compareeach group’s median. P values of less than 0.05 were regarded as statisticallysignificant. The spatial T2 value distribution across the tumour radius at fourdifferent locations from the tumour centre to the outer infiltration zoneadjacent to healthy brain tissue was analysed based on the ROI evaluation. MeanT2 values obtained from these ROIs were modelled using a second-degreepolynomial function. The coefficients of the polynomial were calculated byminimising the sum of the squares of the deviations of the data from the model.Both the original data and the model data were presented in the plots. Thesecond-degree fit accurately follows the basic shape of the data. Higher-degreepolynomial fitting does not improve the results of the model. A higher fit modelvalue (positive or negative) describes the curve’s opening angle. To validatethe model the coefficient of determination (P = 0.0049 – fit model (curvesskewness): GBM –25.02±19.89 ((–54)–10); AA3 –5.57±22.94 ((–51)–47)). TheR2 fit model showed a strong statisticalproof for both subgroup analyses   , 3 and 4±0.15 , 3P = 0.0430 – fit model: IDHw–10.35±16.20 ((–51)–0); IDHm 12.14±21.24 ((–15)–47)). The fit model showed afair statistical proof for both subgroup analyses divided by mutationalstate   –5.P<0.001) and the WHO grade(P<0.001). Patients with AA3 (43.11±11.22 years) are youngerthan patients with GBM (62.14±14.71 years), while IDHw is associated with anolder age than IDHm. There were 17 female and 24 male subjects included. MGMTpromotor methylation prevails significantly in IDHm gliomas(P<0.001). Regarding the WHO grade, no significantdifferences have been reported (P = 0.052). The tumourlocalisation does not correlate significantly with the WHO grade(P = 0.510) or the IDH status (P = 0.061).There was no significant difference between the maximal axial tumour diameter inthe T1/T1 contrast enhanced sequences from AA3 (43.58±20.11 (range 17–81) mm)and GBM (40.91±17.16 (range 18–86) mm); P = 0.811).Nevertheless, GBMs (20.55±6.04 (range 10–36) mm) had a larger diameter in theT2/FLAIR sequences, potentially reflecting the oedema/infiltration zone than AA3(15.84±6.14 (range 8–29) mm; P = 0.012). Assorting by the IDHstatus revealed that IDHm gliomas had a significantly larger tumour diameter(P = 0.025) but a significantly smaller oedema/infiltrationzone (P = 0.011) than IDHw gliomas. All other patientcharacteristics, distributions and values are provided in Overall, there is a strong difference in age concerning the IDH status showed that the relaxationtimes acquired by T2 mapping sequences were able to distinguish between IDHw andIDHm WHO grade II and III gliomas, with wildtypes showing lower T2 relaxation times. In another recently published study also by Kern et al. (2020), the groupinvestigated the tumour centre and the tumour periphery showing that valuesdecreased in the periphery. The authors assumed that T2 values may reflect the peripheral tumour cellularactivity as this is the spot of growth and infiltration. The underlying study showed for GBM in comparison to AA3 an increased signaldrawdown from tumour centre to periphery. According to the conclusion of Kernet al., the results of that study might indicate an increased tumour cellularity andwith this an increased aggressiveness and increased occurrence of recurrence, afterresection of GBMs compared to AA3s.The experience and evidence in the literature for HGG regarding mapping techniques islow, although during the past decade mapping techniques are becoming more and morepopular within clinical routine  as it givesthe opportunity to measure the tissue and its signal behaviourquantitatively.5 In this study HGGs were not onlysubdivided into GBM and AA3, but the cohort was further subdivided regarding themutilation state. Peritumoral zones were investigated and in line and according tothe above-mentioned biologically more aggressive HGG wildtypes showed an increaseddrawdown from tumour centre to periphery, similar to GBMs. This may also emphasisethe potential of T2 mapping sequences, for differentiating between HGG molecularsubtypes and might give the possibility for assessing the risk of developing anearly recurrence. To the best of our knowledge, no MRI method is established todefine the peritumoral infiltration zone. The underlying data are emphasisingfurther investigations regarding T2 mapping to define and gain more insight into theperitumoral zone. With this, the extent of tumour resection might be defined moreprecisely, directly influencing tumour burden and patient prognosis.25The WHO classification was revised in 2016 and now includes molecular markerprofiles. The reason for this is that especially IDHw grade II and III gliomas canact like GBMs and have a similar dismal prognosis. Future prospective aims will be to refine the prognostication of mappingrelaxation values to establish values to characterise further the peritumoral zoneand to differentiate between oedema and infiltration.An interesting study by Blystad et al. (2017) analysed the peritumoral oedema ofmalignant gliomas quantitatively also using T1 and T2 mapping sequences. The authorsfound a gradient of relaxation values in the peritumoral oedema closest tocontrast-enhancing parts of the tumour and an increase of the gradients in theoedemas after contrast agent injection.The retrospective and non-blind study design has introduced some detection bias.Another limitation might be the relatively small patient cohort and a missingvolume-based analysis, while this study used a regionally based approach.However, the results of this study are promising and novel. Furthermore,although the sample number is small the statistically calculated validity levelsare sufficient. A further limitation is the use of two different scanners.Nevertheless, although two different types of scanner were used, T2 values ofbrain parenchyma are not affected significantly by the field strength.This is one of the first studies investigating T2 mapping sequences to characterisethe tumour centre and periturmoral zone within HGGs. The results show that theperitumoral relaxation behaviour differs from GBMs to those of AA3s. Interestingly,when subdivided by their molecular IDH profile, the more aggressive IDHw show asimilar relaxation behaviour as GBMs. These results emphasise the potential of T2mapping techniques to reflect the tissue composition of HGGs. With this adifferentiation between active tumour cells and vasogenic oedema in the peritumoralzone might be possible. The results of this study add information to the ongoingdiscussion on how to facilitate more accurate resection margins potentiallydecreasing early recurrence.The author(s) declared no potential conflicts of interest with respect to theresearch, authorship, and/or publication of this article.


Solid organ transplant recipients, due to the administration of post-transplant immunosuppressive therapies, are at greater risk of viral reactivation episodes, mainly from herpes viruses, including varicella-zoster virus (VZV). The aim of this pilot study was to develop functional immunological assays (VZV-ELISpot) for the quantification and characterization of the VZV-specific effector-memory and central-memory responses in healthy subjects and transplanted patients. Glycoprotein gE and immediate-early 63 (IE-63) were used as antigens for in vitro stimulation. VZV-seropositive healthy subjects showed higher responses in respect to seronegative subjects. Even if differences were observed between VZV-seropositive healthy subjects and transplanted subjects at pre-transplant, the VZV-specific T-cell response was reduced at 60 days after transplant, mainly for the high level of immunosuppression. Phenotypical characterization revealed that response against VZV was mainly mediated by CD4 T cells. The results obtained in this study might be useful for the definition of personalized follow-up of the transplanted patients, providing useful information on the status of the patient potentially at risk of viral reactivation or other opportunistic infections. Varicella-zoster virus (VZV) belongs to the α-herpesvirus family, the causative agent of Chickenpox (a highly contagious exanthematic disease that occurs mainly at a pediatric age and that develops because of primary infection) and shingles, also called zoster, caused by the reactivation of the virus that remains latent in the dorsal sensory ganglia. Knowing the condition of a patient’s immune system is fundamental to understand the risk of severe viral infections and their progression. The risk of VZV reactivation or impaired VZV control, as observed with other herpesviruses, can be detected, especially in patients with immune system deficiencies. Rondaan et al. demonstrated a decreased cellular immunity to VZV in systemic lupus erythematosus patients , while oIn our pilot study, we characterized two immunological approaches for the quantification of VZV-specific T-cell response. Specifically, two methods for the quantification of VZV-specific T cells, known as ex vivo or standard ELISpot assay and cultured ELISpot assay, were designed. Overall, they allow, with a good estimation, the identification of effector memory (Tem) and central memory (Tcm) T-cells, respectively. In detail, ex vivo ELIspot assay allows the identification of cells with immediate secretion functions that are thought to be mainly Tem cells . From a In a clinical setting, over 90% of adult solid organ transplant (SOT) recipients are seropositive for varicella-zoster virus (VZV). This means that almost the entire adult population has experienced a primary VZV infection. Rates are lower in pediatric transplants ,17. Our Residual stored samples obtained from 35 healthy VZV-seropositive and 7 VZV-seronegative healthy subjects  were used for the setting of immunological assays. Twelve VZV-seropositive kidney transplant recipients  enrolled at the time of transplant and after 60 and 180 days were also considered in the study.The study was performed according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Fondazione IRCCS Policlinico San Matteo (proc no. 38433/2017).Peripheral blood mononuclear cells (PBMCs) were isolated by standard density gradient centrifugation using Lymphoprep  from heparinized whole blood samples. Isolated PBMCs were then cryopreserved in fetal calf serum  supplemented with 10% dimethyl sulfoxide  and preserved in liquid nitrogen. After thawing, viable cells were counted and used in ex vivo and/or cultured ELISpot and intracellular cytokine staining (ICS) assay.For quantification of anti-VZV antibodies in plasma, the automated chemiluminescent microparticle immunoassay system  was used. A positive result for VZV IgG was considered when in the presence of at least 135 U/mL.For the ELISpot set-up, two pools of lyophilized synthetic peptides (15 mers with 11 amino acids overlap) were used, spanning the glycoprotein E  and the Immediate-early protein 63  . Each peptide pool was dissolved in Dimethyl sulfoxide (DMSO) and diluted in an RPMI medium. In each experiment, a final concentration of 0.25 μg/mL of each peptide was used. Human interferon-gamma (IFN-y) ELISpot kits  and MultiScreen-IP membrane-bottomed 96-well plates  were used for standard ELISpot assays and for cultured ELIspot assays according to the previous protocols, with some modifications ,19.2 humidified atmosphere for 24 h. After washing, the plates were incubated at 37 °C for 90 min with a biotinylated IFN-γ detection antibody. The plates were then washed, and streptavidin-alkaline phosphatase conjugate was added. The plates were incubated at 37 °C in a 5% CO2 atmosphere for 60 min. After washing, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) was added for 20 min at room temperature. The wells were then washed several times under running water and air-dried overnight. Spots were counted by using an automated AID ELISPOT reader system . The mean number of spots from duplicate were adjusted to 1 × 106 PBMC. The net spots per million PBMC was calculated by subtracting the number of spots in response to negative control from the number of spots in response to the corresponding antigen.For ex vivo ELISpot assays, the plates were coated overnight with a monoclonal capture antibody against IFNγ and stored at 4 °C. After washing with PBS, the plates were blocked with culture medium  ) for 2 h at room temperature. Cells were plated in duplicate (/100 μL per well) and stimulated with the corresponding antigens or with phytohemagglutinin  or with medium alone (negative control) and incubated at 37 °C in a 5% CO2 humidified atmosphere. Then, PBMC were cultured (1 × 106 cells/mL per well) in 24-well plates  and stimulated with gE or IE63 pools or medium alone. The plates were incubated for 10 days. On days 3 and 7, 500 µL of the medium was removed and replaced with 500 µL of cultured medium containing 20 IU/mL of recombinant human interleukin-2 . On day 11, cells from each well were washed, resuspended in the culture medium at a concentration of 1 × 106/mL and kept overnight at 37 °C humidified atmosphere 5% CO2 before the ELISpot procedure, as previously described.For the cultured ELISpot assays, PBMC were resuspended in a cultured medium and rested overnight at 37 °C in a 5% CO6 cells were transferred to a 96-well round-bottom plate in 100 µL culture medium with fetal calf serum in the presence of the corresponding peptide pool or SEB or culture medium alone. Following 1-h incubation at 37 °C in a humidified 5% CO2, brefeldin A (Sigma-Aldrich) at a final concentration of 10 ug/mL was added. After overnight incubation, cells were washed in phosphate-buffered saline (PBS) ethylenediaminetetraacetic acid (EDTA) 2mM and incubated with the Live/Dead Fixable Far Red Dead Cell Stain Kit  for 30 min at 4 °C. Cells were then washed with PBS, fixed and permeabilized with BD Cytofix/Cytoperm  according to the manufacturer’s instructions for intracellular staining with the following monoclonal antibodies: IFNγ-FITC, CD4-ECD, CD8-PC7, CD3-Pe-Cy5 . Finally, the cells were resuspended in 4% paraformaldehyde and analyzed with a Navios flow cytometer , obtaining the percentages of IFN-γ secreting lymphocyte cell responses and allowing phenotypical discrimination of individual cytokine-producing cellsThe available number of PBMC allowed further analysis of eight samples by flow cytometry. In detail, 0.5 × 10p < 0.05 were considered statistically significant.For each condition tested in duplicate, the mean of spots obtained was calculated, adjusting the number to 1 million PBMC. Net spots/million PBMC was obtained by subtracting the number of spots in wells with culture medium only from the number of spots in wells from each VZV peptide pool. The proliferation index (PI) for each tested condition was also calculated in cultured ELISpot assay, as the ratio of the number of cells proliferated during the 12 days of culture stimulated with PHA or gE or IE63 and the number of cells proliferated during the same period in the presence of the culture medium only. All results were shown in terms of net spots/million PBMC multiplied by the PI (net spots /million PBMC×PI). For the comparison of two groups, quantitative variables were analyzed using the Mann–Whitney U test. The analysis of the variations with time of antigen-specific response in transplant recipients was performed with the Friedman test for repeated measures. Finally, the correlation between T-cell responses and the antibody titer was performed using Spearman tests, as well as the correlation between responses measured by ex vivo and cultured ELISpot against gE and IE63 specific T-cell response. All analyses were performed using GraphPad Prism software . Results with p = 0.0002) and was 0.0 (IQR 0–10) net spot/million PBMC in seronegative controls. IE63-specific T-cell response was 48 (IQR 20–95) and 3 (IQR 0–18) net spot/million PBMC in seropositive and seronegative, respectively (p = 0.007)  net spot/million PBMC in seropositive subjects (= 0.007) . The cutp = 0.0033), while median IE63-specific T cell response was 67.5 and 6 net spot/million PBMC, respectively (p = 0.1014)  . The cut2 = 0.01; p = 0.6880), as well as between VZV-specific IgG antibody titer and IE63-specific T-cell response measured by ex vivo ELISpot  in the 26 subjects tested (data not shown). Similarity no correlation was observed between VZV-specific IgG antibody titer and gE specific T cell response , as well as between VZV-specific IgG antibody titer and IE63-specific T cell response  when measured by cultured ELISpot assay (data not shown). Moreover, no correlation was observed between the two assays when gE specific T-cell response  and IE-63 T-cell response  were measured (data not shown).No correlation was observed between IgG VZV-specific antibody titer and gE specific T-cell response measured by ex vivo ELISpot (r+ or CD8+ T-cells subsets was investigated in seven VZV-seropositive by ICS for IFNγ after overnight stimulation with cognate antigens. The median percentage of CD8+ T-cells specific for gE antigen was 0.175 (IQR 0.097–0.225), while the median percentage of CD4+ T-cells specific for gE antigen was 0.330 (IQR 0.177–0.505). The median percentage of CD8+ T-cells specific for IE63 antigen was 0.155 (IQR 0.047–0.345), while the median percentage of CD4+ T-cells specific for IE63 antigen was 0.250 (IQR 0.127–0.562). (The distribution of VZV-specific T cells among CD4–0.562). . An examp = 0.6693), a trend of significance was observed in terms of median gE-specific T-cell response (p = 0.0569). VZV-specific T-cell response measured by cultured ELISpot assay in these patients was not evaluated due to the small amount of PBMC obtained from patients. Twelve kidney transplant recipients were monitored during the first six months post-transplant for VZV-specific T-cell response at 60 and 180 days post-transplant. A decrease of gE-specific T-cell response was observed at day 60 (41 (IQR 11.25–51.25) net spot/million PBMC) (p = 0.0529), which increased significantly at day 180 (115 (IQR 31.88–181.90) net spot/million PBMC; p = 0.0431) (p = 0.0194), respectively  and compared to VZV-specific T-cell response observed in VZV-seropositive healthy volunteers. The median gE-specific T-cell response was 86 (IQR 36–363) net spot/million PBMC, and median IE63-specific T-cell response was 57 (IQR 15–155) net spot/million PBMC. While median IE63-specific T-cell response was not statistically different from that observed in healthy subjects ( 0.0431) a. Similaectively b.This report describes the set-up of an in vitro immunological method for the quantification of VZV-specific T-cell response, using two approaches defined as ex vivo and cultured ELISpot assay that are thought to stimulate mainly T effector memory and T central memory responses, respectively . The firVZV-specific antibody response and T-cell response against VZV antigens measured by ex vivo ELISpot assay were evaluated, and correlation between the two responses was investigated. No correlation was observed between IgG VZV-specific antibody titer and gE or IE63 specific T-cell responses, according to the results obtained by Rondaan et al. . It has We observed that T cell responses against gE and IE63 antigens were not statistically different between healthy controls and transplanted patients. Moreover, in some cases, T-cell response against VZV observed in patients at pre-transplant was higher than that observed in healthy subjects. In this setting, increased production of cytokines is a well-known feature of patients with end-stage renal disease, which is thought to be related to the pro-inflammatory state caused by uremia ,26. We tInterestingly, the decrease of immune response between the day of transplant and 60 days after transplant is not statistically significant, while a significant increase has been detected between day 60 and day 180 but not between day 0 and day 180. This significant increase in terms of VZV-specific T-cell responses was observed at day 180 according to the end of the maximum immunosuppression period due to the normalization of the administered therapy leading to a reconstitution of normal immune function.Even if a reduction of VZV-specific T-cell response was observed after transplant, no episodes of VZV reactivation have been observed in our subsets of transplanted patients. However, four of them (33.3%) developed episodes of Herpes Simplex Virus 1 or Herpes Simplex Virus 2 reactivation in the period of the most intensive immunosuppression. Moreover, it is possible that real cases of VZV reactivation have been underestimated since unreported herpes zoster episodes may be the consequence of an asymptomatic endogenous viral reactivation, as Smetana et al. previously noted to occur in patients receiving renal replacement therapy (RRT) . In thisThe determination of lymphocyte-specific T-cell responses may be a simple method to identify, in healthy subjects and solid transplant patients, the risk of developing opportunistic infections. It may therefore be useful for the stratification of patients according to the risk of reactivation, for the introduction of prophylaxis therapies and for the modulation of immunosuppressive therapy. This immunological monitoring strategy thus represents an important milestone for the personalized medicine perspectives and management of the patient in the post-transplant period.


There are various causes of primary amenorrhea in phenotypically females such as, complete androgen insensitivity syndrome, pure gonadal dysgenesis, 17b-hydroxysteroid dehydrogenase deficiency, or mixed gonadal dysgenesis. Primary amenorrhea in a phenotypically female is commonly encountered in Androgen Insensitivity Syndrome. In patients with Androgen Insensitivity Syndrome, with intraabdominal testis there is high chances of developing testicular tumour, among them Sertoli cell tumour and seminoma being the most common types. Leydig cell tumour in androgen insensitivity syndrome, is very rare and malignant leydig cell tumour is even further rarer. There are few cases reported in the literature of malignant leydig cell tumour with complete androgen insensitivity. Here we are reporting a case of 65 years married elderly patient with malignant leydig cell tumour with complete androgen insensitivity syndrome. Testicular tumors can develop in patients with AIS and that the risk of gonadal malignancies increases as the age increases. Although AIS is diagnosed at puberty in primary amenorrhea with phenotypically female patients, is occasionally diagnosed in an older population also. Complete Androgen Insensitivity Syndrome (CAIS) is associated with abnormal testicular development with an increased risk of germcell malignancy. As the age advances the risk of malignancy increases.3 Sertoli-Leydig cell tumours are very rare gonadal tumours, majority being benign.Androgen Insensitivity Syndrome (AIS) or testicular feminization is a sex-linked recessive inherited disorder caused by a mutation of the androgen receptor gene located at Xq11-q12J Patients with AIS have abnormal descend of the testes, histologically revealing solid immature tubules and markedly decreased or absent germ cells. The gene (containing 8 exons) for the androgen receptors is located on the long arm of X chromosome.There are reports of various types of tumours in AIS patients. Though Leydig cell tumour is the most common type of pure sex cord-stromal tumour of the testis in the general population, Leydig cell tumour in patients with AIS is extremely rare. We hereby reporting a case of malignant Leydig cell tumour in a 65-year-old patient with complete AIS.A 65 years old patient was referred to Bir Hospital, Kathmandu Nepal from the local hospital in Hetauda, Nepal with huge inguino-scrotal swellings, which became tense and painful for last 1 month after the attempt of aspiration from the swelling. Patient is married for the last 35 years, though having no children and with primary amenorrhea but has not consulted doctors irrespective of the symptoms. On physical examination, patient is 165 cm tall and 55 kg weight with normal breast and sparse axillary and pubic hair development. The external genitalia appeared to be those of a normal female. The vagina was shortened and blind. There was no uterine cervix observed. There was no physical evidence of virilisation.Routine haematological and biochemical investigations were within normal limits. Ultrasound examinations of the abdomen, pelvis and perineum revealed bilateral testis. There was a mixed echoic lesion with mixed vascularity noted in bilateral scrotal sac. However, defect was not appreciated likely inguinoscrotal hernia. Patient has two sisters and two brothers; none of them had similar abnormalities and they are married with children. Hormonal profile and tumour markers for the patient evaluated showing: serum LDH/Alpha Feto-protein/ serum beta HCG: WNL, testosterone: 0.92 nmol/L , progesterone: 0.22 ngm/ml , estradiol: 26.35 pgm/ml .CT scan abdomen and pelvis showed undescended right testis, heterogeneous enhancing enlarged left testis likely due to inflammatory causes- orchiepididymitis, rudimentary prostate.Cytogenetic analysis demonstrated a male karyotype . Based on these data, patient was diagnosed as Complete Androgen Insensitivity Syndrome (CAIS).Patient was taken for surgical exploration, which revealed large 25 cm × 20 cm size swelling with haemorrhagic fluids in the sac with firm irregular mass in sac involving the left testis. Right testis was within the right inguinal canal.The final pathological diagnosis of the tumour was malignant leydig cell tumour of the left testis. The extirpated left gonadal tumors was brown coloured, single piece of capsulated globular tissue measuring 17cm × 1.5 cm × 8 cm, testis: 7.5 cm × 6 cm × 3.5 cm. Cut section showing, tan white mass with areas of haemorrhages measuring 7 cm × 4 cm × 3 cm . HistoloPost operatives days were uneventful and the patient was discharged on the tenth postoperative day. Patient was asked to follow up on the subsequent OPD visits for further evaluation and the need for adjuvant chemotherapy but lost on follow up there after.4 It occurs due to a defect in androgen receptor function, causing peripheral androgen resistance. It is characterized by a normal female phenotype however with a male karyotype. There is presence of female breast development, although no axillary or pubic hairs and absence of mullerian duct derivatives. The external genitalia appear normal with usually shortened and blind end varying length of the vagina. The cervix and uterus are absent. The undescended testes may lie along the normal course of testicular descent like in abdominal cavity, the inguinal canal or the labia majora.CAIS is a rare pathologic condition, incidence ranging from 1/60,000 to 1/20,000 births.5 These testes should be removed by either laparotomy or preferably using laparoscopy in case of intra-abdominal testes. Leydig cell tumour, very rare in patients with AIS, is though the most common type of testicular pure sex cord-stromal tumour in the general population. Commonly seen tumours in AIS are Sertoli cell tumour and germ cell tumours. Rutgers and Scully has reported one case of Leydig cell tumour in patients with AIS in the series of 43 cases.6 They reported 1.5 cm in diameter Leydig cell tumour without crystals of Reinke in a 49-year-old patient. In the largest series of Leydig cell tumours of the testis by Kim et al, reported the prognosis and characteristics of this malignant tumor.7 They were followed-up from 2 months to 22 years (average 4 years). When they compared survivors and non-survivors, showed that the factors associated with a decreased survival were increasing patient age, larger tumour size, infiltrative margin, spread beyond the testis, invasion to blood vessels or lymphatics, greater degree of cellular atypia, necrosis, and a higher mitotic rate. Our case had four out of eight above features described.The estimated risk of malignancy the testis is 5–10% until 25 years of age and increases up to reach 33% at age 50 years.Leydig cell tumour is rare in-patient with AIS. Malignant Leydig cell tumour is further rare. Our case represents the identification of a rare tumour in a minority group of people in resource-limited country like in Nepal, which is genetically and histologically proven.


Casson fluid is greater than the thermal efficiency of Casson fluid.The rheological model for yield stress exhibiting fluid and the basic laws for fluid flow and transport of heat and mass are used for the formulation of problems associated with the enhancement of heat and mass due to dispersion of nanoparticles in Casson. The heat and mass transfer obey non-Fourier’s laws and the generalized Fick’s law, respectively. Model problems are incorporated by thermal relaxation times for heat and mass. Transfer of heat energy and relaxation time are inversely proportional, and the same is the case for mass transport and concentration relaxation time. A porous medium force is responsible for controlling the momentum thickness. The yield stress parameter and diffusion of momentum in Casson fluid are noticed to be inversely proportional with each other. The concentration gradient enhances the energy transfer, and temperature gradient causes an enhancement diffusion of solute in Casson fluid. FEM provides convergent solutions. The relaxation time phenomenon is responsible for the restoration of thermal and solutal changes. Due to that, the thermal and solutal equilibrium states can be restored. The phenomenon of yield stress is responsible for controlling the momentum boundary layer thickness. A porous medium exerts a retarding force on the flow, and therefore, a deceleration in flow is observed. The thermal efficiency of  The diverse rheological behavior of non-Newtonian fluid motivated researchers to propose diverse rheological models. Each model captures some specific rheological features. Casson rheological model is a non-Newtonian rheological model and captures yield stress rheological features. This means, to some extent, Casson fluid behaves like a solid and, after some specific value of applied stress, it starts behaving like a fluid. This specific value of applied stress is called yield stress. The simplest rheological model for the incompressible flow of Casson fluid is given byτ=−PI+–(7) in dimensionless form can be changed via the following change of variable:Hence, one obtains:The associated boundary conditions are dimensionalized, and hence, one obtains:Derivatives concerning The skin friction and Nusselt and Sherwood numbers are obtained asThermophysical properties are tabulated in The different numerical schemes can be applied for the solution of Equations (8)–(12). However, FEM is powerful and provides the most accurate numerical solutions. The main steps are mentioned below.Step 1: Residuals are multiplied by weight functions and are integrated over the typical element.Linear weight and shape functions are given byThe dependent unknowns are approximated over the element Step 2: The weighted integrals are evaluated only for second-order linear terms to obtain weak forms of residuals.Step 3: The weak forms are approximated via Galerkin approximations.Step 4: Stiffness elements are derived, and the stiffness matrix is constructed under the assembly procedure.Step 5: The nonlinear system is linearized.The nonlinear system is linearized by Picard linearization proposed asStep 6: A grid-independent study is performed.The numerical solutions are also checked to be grid independent. This mesh-free analysis is recorded in Step-7: Results are validatedThe present modeled problems can be reduced to the case of already published work by Aninasaun et al.  when φ1=n et al. . The comThe yield stress rheological model, non-Fourier law of heat conduction, and generalized Fick’s law are simultaneously used in the formulation of problems dealing with the simultaneous transfer of heat energy and mass in Casson fluid with hybrid nanoparticles over a vertical surface. The buoyancy forces due to the density differences under Boussinesq approximations are considered of a significant order of magnitude, and Lorentz force is measured from Ohm’s law when the magnetic Reynolds number is small. The numerical experiments with three samples of parametric values are performed. The simulations are visualized, and the outcomes are displayed in the form of graphs and numerical data. The mesh-free analysis is performed, and the results are validated. A detailed discussion about visualized outcomes is provided below.Fluid particles motion and magnetic field: The Lorentz force is incorporated in the momentum equation through Ohm’s law and the term involved Hartmann number in the dimensionless equation (Equation (8)). This dimensionless number luid see .Fluid particles motion and resistance by the porous medium: The parameter K is called the porous medium parameter, and its variation through increasing values ions see . It is aFluid particle motion and buoyancy force: The parameters flow see , whereasTemperature of fluid particles and intensity of magnetic field: The M is due to Joule heating (Ohmic heating) and determines the impact of Ohmic dissipation on the temperature of the fluid. This dissipated heat raises the kinetic energy of the fluid particles, and therefore, their temperature increases. This effect can be noticed in Temperature of fluid particles and viscous dissipation: The friction among the particles of fluid causes the dissipation of heat which is equal to the rate at which the work is performed by friction force. This effect is called viscous dissipation. It is noticed during simulations that the inclusion of hybrid nanostructures .The thermal relaxation time is responsible for decreasing the temperature of fluid. This is because of the ability of the fluid to restore thermal changes in order to attain thermal equilibrium.Future aspects: In this study,


Control animals were kept in normoxia and received infusions or injections of 0.9% NaCl. After 6 h of hypoxia, LV inotropic function was maintained with NaCl injection but decreased significantly with NaCl infusion. Adrenergic blockade induced a similar LV depression when fluid load was low, but did not further deteriorate LV depression after 6 h of infusion. Reduced fluid load also attenuated pulmonary injury after 6 h of hypoxia. This might be due to an effective fluid drainage into the pleural space. Adrenergic blockade could not prevent PE. In general, increased fluid load and impaired LV inotropic function promote the development of PE in acute hypoxia. The main physiologic conclusion from this study is that fluid reduction under hypoxic conditions has a protective effect on cardiopulmonary function. Consequently, appropriate fluid management has particular importance to subjects in hypoxic conditions.Acute normobaric hypoxia may induce pulmonary injury with edema (PE) and inflammation. Hypoxia is accompanied by sympathetic activation. As both acute hypoxia and high plasma catecholamine levels may elicit PE, we had originally expected that adrenergic blockade may attenuate the severity of hypoxic pulmonary injury. In particular, we investigated whether administration of drugs with reduced fluid load would be beneficial with respect to both cardiocirculatory and pulmonary functions in acute hypoxia. Rats were exposed to normobaric hypoxia (10% O Exposure to acute hypoxia may induce pulmonary injury characterized by pulmonary edema (PE). This is a typical complication in travelers to high altitude called high-altitude PE (HAPE). Although hypoxia-induced PE such as HAPE is considered a hydrostatic edema , 29, it Hypoxia is associated with increased sympathetic activity , 30. HigA previous study on rats  showed tTherefore, we designed the present study to investigate some questions related to the importance of additional fluid load and sympathetic activity for cardiopulmonary function under conditions of acute hypoxia. We compared increased fluid load (infusion at 1 ml/h) with reduced fluid load achieved by a single injection with low fluid volume prior to the exposure to hypoxia. The role of fluid load is studied with and without adrenergic blockade. Adrenergic blockade serves as a model of suppression of hypoxia-induced sympathetic effects. To the best of our knowledge, this is the first study directly comparing effects of low versus high fluid load and drug administration in acute hypoxia. Specifically, we investigated whether (a) increased fluid load aggravates cardiocirculatory dysfunction and hypoxia-induced pulmonary injury and (b) adrenergic blockade may prevent or attenuate pulmonary injury when additional fluid load is low.All experiments were performed on 147 female Sprague–Dawley rats supplied by Charles River . The body weight was 225 ± 23 g at the beginning of the study corresponding to an age of about 12 weeks. All animal protocols were approved by the Federal State Agency. The experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and with the “European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes” .Animals were exposed to normoxia (N) or normobaric hypoxia (H) for 1.5 or 6 h. For exposure to hypoxia, the animals were placed into a hypoxic chamber sized 65 × 105 × 50 cm. The gas mixture in the chamber contained 10% oxygen in nitrogen. Special equipment prevented penetration of ambient air during manipulations on the animals, thus keeping the oxygen concentration in the chamber stable at 10 ± 0.5%. The hypoxia-exposed animals were treated with adrenergic blockers or with 0.9% sodium chloride (NaCl) solution. We used the α-adrenergic blocker prazosin (PZ), the β-adrenergic blocker propranolol (PR), or a combination of both , all drugs were obtained from Sigma-Aldrich .The fluid was administered either by i.v. injection at the start of the experiment (inj) or i.v. infusion over the total experimental time (inf). The infusion rate was 1 ml/h, while the amount of injection fluid was 0.5 ml. The drug doses were chosen according to previous experiments . They we−1 and remained in anesthesia until the end of the experiment. Exposure to hypoxic environment started immediately after this initial operation.For fluid application, the animals were anesthetized, and the left jugular vein was prepared. Injection was given within 15 s into the vein, which was ligated afterwards. For infusion, a catheter  was inserted into the vein, and the fluid was administered with an automatic pump . For experiments over 6 h, this operation was performed in 2% isoflurane anesthesia. These animals woke up after injection or catheter insertion and moved freely with access to tap water and rat chow diet . Animals allocated to 1.5 h of treatment were anesthetized with an intraperitoneal (i.p.) injection of thiopental sodium  80 mg kg−1, i.p.) and started about 30–40 min before the end of the exposure time. The animals were tracheotomized, and a polyethylene cannula was placed in the trachea. RV and LV were catheterized with Millar®  ultraminiature catheter pressure transducers  to measAnimals were sacrificed by exsanguination from the abdominal aorta. In the next step, we opened the thoracic wall and collected pleural fluid (PF). After ligation of the right main bronchus, a bronchoalveolar lavage (BAL) was performed two times consecutively with 3 ml 0.9% NaCl each. The fluid was instilled via the tracheal cannula into the left lung and withdrawn immediately. The recovery rate was about 90% on average. Heart and right lung were then rapidly excised. Tissue samples of the cardiac apex and of the upper and lower lobes of the right lung were fixated in formalin for histological analysis. The blood was centrifuged for 10 min at 2100 rpm. Serum, BAL fluid, and further samples of lung tissue were frozen and stored at -80 °C for further analyses.The formalin-fixated lung tissue samples were embedded in paraffin, sliced, and stained with hematoxylin–eosin (HE). Histological assessments were done by two independent investigators (PG and KS), who were blinded towards the treatment group. In the lungs, they evaluated pulmonary edema (PE), inflammation, and vascular hypertrophy. For a detailed quantification of PE, about 16–24 single slices from each animal (8–12 slices each from the upper and lower lung lobe) were evaluated. First, PE severity in each area of the section was scored with 0 (absent), 1 (mild), 2 (moderate), and 3 (severe). As no differences between upper and lower lung lobes were apparent, values from all sections of the two lobes were pooled. The pulmonary edema index (PEI) was calculated by cumulating the products of edema score and proportionate area of each part of all slices ) Finally, the results of this study cannot explain the reasons for hypoxic depression of LV inotropic and lusitropic functions and its aggravation by increase fluid load. Impaired LV energy metabolism and a contribution of ventricular stretch receptors are two mechanisms supposed to be involved, but further research on causal relations is necessary.Acute hypoxia over 6 h combined with increased fluid load depressed LV inotropic and lusitropic function and induced pulmonary injury characterized by inflammation and edema. Reduced fluid load attenuated these cardiac and pulmonary effects of hypoxia.Sympathetic activation in hypoxia was mild and could only partially compensate for hypoxia-induced LV depression. Combination of hypoxia with increased fluid load even compromises sympathetic activation or sympathetic effects on the heart. Ventricular stretch reflexes or hypoxic impairment of myocardial energy metabolism might be possible explanations, but further studies are necessary.Factors increasing pulmonary perfusion or pulmonary capillary pressure promote PE formation. Sympathetic activation plays a minor role for PE formation in acute hypoxia. However, even with low fluid load, adrenergic blockade cannot prevent PE. This may be attributed to the differential effects of the various factors on the delicate balance between pro- and anti-edematous mechanisms.Under acute hypoxic conditions, appropriate fluid management has particular importance for adequate cardiopulmonary function.


The first error regards Table S1. Unfortunately, we provided an incorrect version of the table that contains incorrect references. The table has been replaced online. Second, we regret not including NIH training grant information for Britta Rued. Britta Rued was supported by the grant while she was collecting data and generating the manuscript. The grant identification number is 5K12 GM139186/NIGMS/NIH.Volume 12, no. 2, e02688-20, 2021,


Molecular organic fluorophores are currently used in organic light-emitting diodes, though non-emissive triplet excitons generated in devices incorporating conventional fluorophores limit the efficiency. This limit can be overcome in materials that have intramolecular charge-transfer excitonic states and associated small singlet-triplet energy separations; triplets can then be converted to emissive singlet excitons resulting in efficient delayed fluorescence. However, the mechanistic details of the spin interconversion have not yet been fully resolved. We report transient electron spin resonance studies that allow direct probing of the spin conversion in a series of delayed fluorescence fluorophores with varying energy gaps between local excitation and charge-transfer triplet states. The observation of distinct triplet signals, unusual in transient electron spin resonance, suggests that multiple triplet states mediate the photophysics for efficient light emission in delayed fluorescence emitters. We reveal that as the energy separation between local excitation and charge-transfer triplet states decreases, spin interconversion changes from a direct, singlet-triplet mechanism to an indirect mechanism involving intermediate states. Despite advances in the design of thermally activated delayed fluorescence (TADF) emitters for devices, the effect of spin interactions is not well understood. Here, the authors report the role of spin-vibronic coupling in TADF organic emitters using transient electron spin resonance spectroscopy. Spin statistics for OLEDs dictate that 75% of electrically-generated excitons populate triplet (spin = 1) states (T1), which, in the case of traditional fluorophores, limits electroluminescence (EL) efficiency since radiative de-excitation of these states to the singlet (spin = 0) ground state (S0) is spin-forbidden8. Current commercial OLED devices employ organometallic chromophores based on iridium and platinum as red and green emitters. In such materials, enhanced spin–orbit coupling from the heavy element promotes direct emission from triplet excitons (phosphorescence), allowing up to 100% internal quantum efficiency (IQE) for EL11. Blue emitters based on the same approach have remained elusive, particularly because the long lifetimes of high-energy triplet excitons lead to OLED degradation13. Consequently, blue OLEDs typically employ organic fluorophores, with an enhancement of EL IQE achieved using triplet-triplet annihilation , which can harvest up to half of the triplet excitons for light emission via singlets16. TTA-based OLEDs can in principle achieve up to 62.5% EL IQE. Within this context, TADF emitters present an alternative approach to achieving 100% EL IQE in devices, thereby offering a step-change in performance for blue OLED technologies18.Thermally activated delayed fluorescence (TADF) organic molecules convert electricity to light more efficiently than traditional organic fluorophores, making them attractive materials for organic light-emitting diodes (OLEDs)1 → S1 → S0) is activated by ambient thermal energy. There is general agreement that TADF emitters should be designed to have small singlet-triplet exchange energies that promote rapid forward and reverse intersystem crossing (ISC) between singlet and triplet excitons; such small exchange energies can be engineered by spatial separation of the highest occupied and lowest unoccupied molecular orbitals (HOMO and LUMO)6. However, there is no agreement on the spin interactions and spin-flip mechanisms responsible for the singlet-triplet interconversion in fluorophores exhibiting TADF with intramolecular charge-transfer (CT) states22. Typical TADF emitters have low-lying singlet and triplet excitonic states with mixed local excitation (LE) and intramolecular CT character23. Emission from the predominantly CT singlet excitonic state (1CT) via prompt or delayed fluorescence is desirable. Therefore, the triplet excitons populating either CT or local excitation states (3CT and 3LE) must be efficiently converted to 1CT. ISC between the singlet and triplet manifolds of fluorophores can be driven by a spin-orbit coupling (SOC) mediated spin conversion, whereby an orbital transition between two states generates a torque capable of flipping an electron’s spin. Direct SOC (dependent only on the electronic character of the coupled states) will in general be suppressed between a singlet and triplet state if they possess similar orbital character (El Sayed’s Rule)25. Therefore, in order to achieve singlet-triplet ISC between two electronic states of the same orbital character (e.g. 1CT to 3CT) additional processes must occur, which are dependent on both the electronic character and nuclear coordinates of the coupled states. However, the presence of a significant change in orbital character upon spin-flip (e.g. 1CT to 3LE) is not in itself sufficient to guarantee rapid ISC and, furthermore, it has been shown that direct SOC alone cannot explain high rates of ISC observed28. Vibration-mediated SOC has been suggested to explain this discrepancy, and also to facilitate singlet-triplet conversion between states of similar orbital character29. Vibrational contributions from an additional, or ‘intermediate’, triplet state give rise to the ‘spin-vibronic mechanism’ for the efficient conversion of dark triplet excitons to emissive singlet excitons (reverse ISC) in TADF emitters32. Revealing the character and formation dynamics of intermediate triplet states is challenging, although critical to understanding the TADF mechanism34.In TADF devices, indirect emission from dark triplet states via bright singlet excitons  it can only explicitly probe forward ISC. However, assuming microscopic reversibility, the triplet exciton formation dynamics revealed are applicable to TADF-relevant reverse ISC. We have previously shown using trESR that spin-orbit coupling interactions mediate ISC for two benchmark TADF molecules: 1,2,3,5-tetrakis(carbazol-9-yl)-4,6-dicyanobenzene (4CzIPN) and 1,2-bis(carbazol-9-yl)-4,5-dicyanobenzene (2CzPN)20. The trESR data suggested a dynamic picture of SOC-mediated ISC involving vibrational modes, although the excited-state ordering and coupling requirements for efficient ISC were not established. To reveal these requirements, here we have conducted trESR studies on a series of ten molecular organic fluorophores exhibiting delayed fluorescence with varying 3LE–3CT energy gaps; this allows us to probe the spin-vibronic mechanism directly and resolve the nature of intermediate triplet states. TrESR gives conclusive evidence that 3LE can be populated from the singlet manifold by the vibrational SOC ISC mechanism. However, when the 3LE–3CT gap is sufficiently small, trESR reveals that additional spin conversion pathways emerge, and 3CT is populated via the spin-vibronic ISC mechanism because of enhanced 3LE–3CT coupling. TrESR can crucially resolve these different ISC mechanisms, which are influenced by difficult to predict molecular properties beyond the excited-state character and energetics measured with optical spectroscopy. Our spin-sensitive measurements reveal direct evidence that distinct triplet excitonic states (T1 + T2) mediate the spin conversion responsible for delayed fluorescence. These findings uncover the fundamental mechanism of the spin-vibronic ISC pathway relevant in TADF emitters, providing detail on the nature of the process by probing distinct yet vibronically-accessible 3LE and 3CT states.Here we use transient electron spin resonance (trESR) spectroscopy to study the role of spin-orbit coupling and vibrational perturbations in ISC for TADF. While optical spectroscopy has proved a powerful tool to examine the photophysics and spin-conversion rates in TADFtert-butyl-9H-carbazol-9-yl)diphenyl sulfone (DTCz-DPS) core structure were synthesised. We employed inductively electron-withdrawing fluorine substituents on a phenyl group linking carbazolyl and sulfone moieties to lower the LUMO energy of the DTCz-DPS core, resulting in lowered CT state energies as in our previous study50. The three emitters, DTCz-DPS-1, -2, and -3  calculations (Supplementary Table 3LE and 3CT minima (the 3LE–3CT gap), in their optimised geometries, is reduced from DTCz-DPS-1 to -3 , which is determined by the nature of the triplet formation (ISC) process. The magnetic field positions of the positive (absorptive) and negative (emissive) peaks in the trESR signal  are determined by the ZFS parameters, D and E  of the spin-polarised trESR signal. Following photoexcitation, the all-organic emitters have typically low triplet yield and hence data acquisition times of 4–12 h were required for each trESR spectrum.For the trESR measurements, pulsed laser excitation (355 nm) and cryogenic temperatures (30 K) were used to generate long-lived, molecular triplet excitons in dilute (500 μM), deoxygenated and flash-frozen toluene solutions. A variable applied magnetic field (200–500 mT) induced Zeeman energy separation of the triplet sublevels from the zero-field splitting (ZFS) eigenstates . The ZFS parameters , the position of the half-field transition  and has a spin-polarisation pattern that indicates relative overpopulation of Tx and Ty . A reduced magnitude of D of this scale typically indicates an increased spatial separation of interacting spins; therefore, we attribute the narrow signal to the delocalised 3CT51.Each emitter exhibited spin-polarised trESR signals with lifetimes of several microseconds, throughout which the spectral shapes were conserved. The trESR signal of DTCz-DPS-1 Fig.  has a 6- -3 Fig.  show spi -3 Fig.  matches 3LE is assigned to the broad triplet signal. By subtracting the 3LE contribution from the total DTCz-DPS-3 trESR spectrum a narrower triplet signal ascribed to 3CT is obtained with Px,y,z = 0.6, 0.4, 0.0, |D | , | E | = 56, 19 mT and a half-field transition peak at 169 mT; the breadth of the DTCz-DPS-3 3CT signal compared to the DTCz-DPS-2 3CT signal is a point to which we will later return. These results show that with decreasing 3LE–3CT gap (from DTCz-DPS-1 to -3), the recorded trESR spectra progress from a relatively pure 3LE signal to one resulting from contributions of both 3LE and 3CT polarisation patterns. The triplet ZFS simulation parameters of all three emitters are shown in Table The trESR signal of DTCz-DPS-3 Fig.  comprise3CT emission, in addition to structured 3LE emission, with decreasing 3LE–3CT gap in the frozen (77 K) toluene phosphorescence spectra . Crucially, both these temperatures are well below the freezing point of toluene (178 K). Therefore, the energetic relaxation of the 1CT states (a common phenomenon undergone by intramolecular CT states in solution) observed when comparing the photoluminescence (PL) in frozen (77 K) and solution (295 K) toluene  in deoxygenated toluene  were calculated for each 3LE and 3CT , or by replacing the tertiary butyl groups with additional carbazole units . The 3LE–3CT gap was decreased by fluorination on going from Cz-DPS-1 to −2 and from 3Cz-DPS-1 to -2; we observed with decreasing gap: (a) emergence of the 3CT trESR signals 1CT and 3LE;Direct spin–orbit coupling between (II)1CT and 3LE;Vibrational spin–orbit coupling between (III)1CT and 3CT via intermediate 3LE.Spin-vibronic coupling between TADF facilitated by spin-orbit coupling, as described in the ‘Introduction’, can be interpreted within the following framework of ISC mechanisms1CT–3LE transitions depend on the nuclear degree of freedom, 3CT and 3LE, equilibrating their population; 3LE is then coupled to 1CT via either (I) or (II)30. 3LE is considered an intermediate in the sequence of state formation: 1CT–3LE–3CT  is assigned to modes associated with the donor–acceptor linker61. These three ISC processes are expressed together by the sum of their first non-zero terms:26. The vibronic coupling between two states generally increases as the energy gap between the states decreases and hence mechanism (III) only significantly contributes to the full spin–orbit interaction if the 3LE–3CT gap (ΔG) is sufficiently small (<0.5 eV). Furthermore, orbital and vibrational mode-dependent mechanisms are determined by the geometry of a molecule and differently affect ISC mechanisms (I), (II), and (III)26. TrESR polarisation patterns are fingerprints for the relative spin population of the triplet ZFS sublevels , which are also related to the molecular geometry62—Fig. 3LE ZFS axes are related to the molecular axes  in DTCz-DPS-1, -2, and -3. These relationships mean that triplet formation by each ISC mechanism will yield a particular trESR polarisation pattern that can be distinguished by the ISC mechanism’s orbital and/or vibrational mode dependence on molecular geometry.Vibrational modes can enhance ISC in (II) because spin–orbit coupling matrix elements for 3CT Fig. . Any ini2 symmetry axis; symmetry does not persist in the delocalised CT states. Under these symmetry conditions the triplet sublevel populations describing the trESR polarisation pattern for each ISC mechanism in a TADF emitter is as follows:(I)2 symmetry axis (x here);Overpopulation along the C(II)x and z here);Overpopulation of the in-plane axes ((III)x and z here) to the out-of-plane (y here) axes.Redistribution of the population from the in-plane  and molecular  axes will be well-aligned. In contrast, the electron density in a 3CT configuration will be delocalised across both the donor and acceptor units  can arise. This additional out-of-plane population causes the difference between the population of Tx,y,z in ISC mechanism (II) and (III)65. The donor–acceptor twist is a fundamental TADF molecular design rule because the spatial separation of HOMO and LUMO is required to reduce singlet-triplet offset and enable reverse ISC. Therefore, we can expect the relationship between molecular structure, ISC mechanism, and trESR pattern to be applicable to many classes of TADF emitters, as well as to donor–acceptor CT-type molecules in general.The difference between ISC mechanisms (II) and (III) is the CT-like transition from 3LE and 3CT in each emitter. The 3LE states of DTCz-DPS-1, -2, and -3  are populated via vibrational SOC with the photoexcited 1CT – mechanism (II) – apparent from the overpopulation of Tx and Tz67. On the other hand, the triplet sublevel corresponding to the out-of-plane molecular axis (Ty) is relatively overpopulated in the 3CT polarisation patterns of DTCz-DPS-2 and -3 (and Cz-DPS-2 and 3Cz-DPS-2). The distinct triplet trESR spectra, therefore, reveal that spin-vibronic coupling between 1CT and 3CT via intermediate 3LE – mechanism (III) – is responsible for 3CT population60. The ability to resolve these ISC mechanisms from one another is a crucial advantage of using trESR; it is not possible to predict how excited states interact based purely on optical measurements of their separation energies or excitation character because additional factors arising from molecular structure affect ISC.Applying the framework outlined above to the triplet sublevel populations from experimental spectra Table , we can 3LE geometries (described in the ‘Methods’) confirms that if direct SOC between 3LE and 1CT – mechanism (I) – were the dominant mechanism we would indeed observe overpopulation predominantly in the 3LE x plane (Supplementary Table 3LE ZFS and in-plane molecular axes (overpopulation in Tx and Tz). A rotation of the in-plane x–z frame upon charge-transfer internal conversion would redistribute population from Tz to Ty, consistent with the experimental observations  compared with ISC (ns)71. Second, in the experiment, the 1CT absorption band was selectively excited  in order to mimic operational OLED conditions where the lowest energy excitonic states are electrically generated , such that 3LE and 3CT are simultaneously populated. It is worth noting that, as TADF development is predominantly focused on the critical task of achieving efficient blue emission, the above outlined spin-vibronic mechanism is described in the context of the energetic regime attainable in blue emitters and shown in Fig. 1CT > 3CT > 3LE22. However, the same principles can be applied to a donor–acceptor emitter exhibiting a different excited-state ordering. We note that the unusual observation of multiple, distinct triplet signals in trESR spectra has also been reported for twisted donor–acceptor triplet photosensitizers, although the mechanism through which both triplets were populated was not rationalised;47 we postulate that the spin-vibronic mechanism may be active in such triplet photosensitizers.To reiterate: our findings show that it is indirect, not direct, coupling of 3CT spectral contribution with decreasing 3LE–3CT gap observed here (and in other systems73) reinforces the direct evidence for the spin-vibronic mechanism from trESR. Furthermore, the emergence of the additional 3CT trESR spectra, reflecting the strength of vibronic coupling, correlates with increased contribution to total emission from delayed fluorescence. However, as the 3LE–3CT gap decreases so does the 3LE–1CT gap and therefore their combined effect on delayed fluorescence cannot be decoupled. We note that the dynamics of excited state couplings are also affected by time- and temperature-dependent molecular reorganisation, as is evident when comparing the 295 K fluorescence with the 77 K fluorescence , are non-interacting and exhibit delayed fluorescence through either the hot-exciton74 or hyperfine pathways75. Our experimental results for the present series of donor–acceptor molecules, supported by computational calculations, show that coupling between the singlet and triplet manifold is facilitated by the spin-vibronic mechanism when 3LE is in energetic proximity (<100 meV) to 3CT. This coupling drives ISC, and therefore TADF-relevant reverse ISC (assuming microscopic reversibility). We have demonstrated that trESR can reveal mechanistic details of ISC, providing a critical, complementary approach to optical spectroscopy methods77 and have established a trESR framework to study the spin-vibronic mechanism in TADF emitters. We have demonstrated that the mechanism for ISC within an emitter can be tuned through chemical modifications; engineering and measuring the spin properties are critical for the informed development of efficient TADF emitters. We have characterised the nature of triplet excitations in the chemically modified set of delayed fluorescence emitters, resolving two distinct triplet excitonic states (T1 and T2) involved in spin-vibronic coupling. This distinction supports the basis for achieving spin-vibronic TADF by engineering distinct yet vibronically-accessible 3LE and 3CT states. As discussed, the mechanism of 3CT population by vibronic coupling with 3LE may find further application in the development of heavy-atom free triplet photosensitizer design47. Tuning spin–orbit coupling in molecular systems and their subsequent trESR signatures, as demonstrated here, is not only of paramount importance to blue OLED development but may also find resonance more broadly in the development of organic materials for light-controlled quantum information and spintronics82.TrESR has been used previously to probe triplet excitons in twisted donor-acceptor emitters that, due to their large TThe emitters were generally synthesised by nuclophilic substitution reactions between the appropriate carbazole derivatives and partially fluorinated derivatives of diphenylsulfone in dimethylsulfoxide in the presence of potassium carbonate, as described in the detail in the 53.Dilute solutions were prepared at 500 μM in toluene in a nitrogen environment. The solutions were contained within 3.8 mm O.D. clear-fused quartz tubes and degassed by 4 freeze-pump-thaw cycles before being flame-sealed. Measurement of transient continuous wave ESR (trESR) was done in the Centre for Advanced ESR (CAESR) in the Department of Chemistry of the University of Oxford, using a Bruker BioSpin EleXSys I E680 at X-band  with an ER 4118X-MD5W resonator. The temperature was controlled with an Oxford Instruments CF935O cryostat under liquid helium flow and an ITC-503S controller. An Opotek Opolete HE355 LASER was used for optical excitation of the samples and it was synchronised to the spectrometer by a Stanford Research DG645 delay generator. Use of a Stanford Research SR560 low-noise preamplifier of the Schottky diode-detected CW Mode signal at 3–300 kHz was in substitute of the video amplifier, following verification against the quadrature mix-down DC-AFC Transient Mode signal of 20 MHz bandwidth setting. Spectra were recorded for between 4 and 12 h. Data was processed with MatLab  and ESR simulations made use of the EasySpin routines83 implemented in the Gaussian 16 programme. The geometry optimisation of the minimum-energy conical intersection between the 3LE and the 3CT was performed with the GRRM17 programme84, which refers to the energy and gradient calculated by the Gaussian 16 programme. 86 implemented in the ORCA 4.2.1 programme. The ZFS parameters of the 3LE and the 3CT were simulated using the DFT PBE0/6–31 G(d) with the spin-spin term calculated from the spin-densities derived from the unrestricted natural orbitals57 and the spin-orbit term calculated by the coupled-perturbed SOC approach58 implemented in the ORCA 4.2.1 programme.The linear-response TD-DFT calculations were performed using the LC-BLYP/6-31 G(d) within the Tamm–Dancoff approximationDilute solutions were prepared at 100 μM in toluene. UV−vis absorption spectra were measured using a Cary 5000UV−vis−NIR spectrophotometer. PL spectra were recorded on a Horiba FL3-2i Fluorometer equipped with a liquid nitrogen attachment in solution at ambient temperature or either as steady-state or gated spectra at 77 K.Dilute solutions were prepared at 100 μM in deoxygenated toluene in a nitrogen environment and subsequently measured in a nitrogen environment. Time-resolved PL spectra were recorded using an electrically-gated intensified charge-coupled device (ICCD) camera (Andor iStar DH740 CCI-010) connected to a calibrated grating spectrograph (Andor SR303i). Pulsed 325 nm photoexcitation was provided at a repetition rate of 1 kHz. Temporal evolution of the PL was obtained by stepping the ICCD gate delay with respect to the excitation pulse. The minimum gate width of the ICCD was 5 ns. Recorded data was subsequently corrected to account for camera sensitivity.Supplementary Information