Although a large amount of data supports the crucial role of endothelial dysfunction (ED) in cardiovascular diseases (CVDs), there is a large bench-to-bedside chasm between basic and clinical research of ED, limiting the implementation of these findings in everyday clinical settings. Hence, it is important to further investigate the pathophysiological mechanisms underlying ED and find modalities that will alleviate its clinical implementation. Adropin, a highly conserved peptide hormone secreted primarily by the liver, recently emerged as an important regulatory component of the vascular endothelium. Specifically, the vasoprotective role of adropin is achieved mainly by affecting endothelial NO synthesis. Thus, in this review, we aimed to summarize the current knowledge regarding the role of adropin in physiological processes and address the protective role of adropin in endothelium with consequent implications to CV pathologies. We focused on data regarding the role of adropin in the clinical setting, with concurrent implications to future clinical use of adropin. Studies suggest that plasma levels of adropin correlate with indices of ED in various pathologies and enhanced disease progression, implying that adropin may serve as a useful biomarker of ED in the upcoming future. On the other hand, despite notable results with respect to therapeutic potential of adropin in preliminary experiments, further well-designed studies are warranted in order to establish if adropin might be beneficial in this setting. Evolving evidence suggests that there are virtually no cardiovascular diseases (CVDs) in which endothelial dysfunction (ED) is not implicated, at least in part . In factAdropin, a highly conserved 76 amino peptide hormone, secreted primarily by the liver, recently emerged as an important regulatory component of the vascular endothelium . Apart fIn this review, we aimed to summarize the current knowledge regarding the role of adropin in physiological process, but in a myriad of pathological processes as well. In particular, we addressed the protective role of adropin in the endothelium, with consequent therapeutic implications for CV pathologies.Encho) gene, which is mainly expressed in the two above-noted organs, especially in areas of the brain involved in metabolic regulation . Won. Won7]. The above-noted regulation of PDH activity seems to be the most important physiological role of adropin. It was demonstrated on adropin-KO mice models that adropin mediates substrate oxidation preference in skeletal muscles . SpecifiAccumulated data indicate that adropin contributes to the modulation of adiposity and metabolism of lipids and glucose in various tissues. Adropin-overexpressing mice challenged with a high-fat diet were protected from body weight gain in the first 6–8 weeks, but not after 3 months of high-fat diet, thus suggesting that adropin overexpression delays but does not completely prevent diet-induced body weight gain . In addiAs adropin is an important cog in the regulation of the glucose and lipid metabolism, the fact that adropin is implicated in the pathophysiology of various metabolic diseases should not be a surprise. Evidence suggests that adropin levels may be affected in diabetes mellitus (DM). Apart from being lower in patients with type 2 DM, lower adropin levels represent a risk factor for ED in these patients . SimilarDiabetic cardiomyopathy (DCM), an under-recognized and lethal complication of diabetes, is commonly defined as cardiomyopathy in patients with diabetes mellitus (DM) in the absence of coronary artery disease, hypertension or dyslipidemia . Among mFurthermore, plasma adropin concentrations significantly correlate with indices of disease severity in patients with obstructive sleep apnea (OSA), as we demonstrated in our previous study . MoreoveFurthermore, in a group of patients with inflammatory bowel disease (IBD), adropin levels were lower in comparison to the control group, and adropin also showed a significant negative correlation with multiple IBD severity scores . The mecIn our previous study conducted on a population of patients undergoing hemodialysis, adropin levels were significantly lower in comparison to controls . MoreoveOverall, although adropin is implicated in a myriad of different pathologies, it seems that all of these have the same denominator—endothelial dysfunction .Finally, adropin seems to be a missing link explaining the higher cardiovascular risk observed in patients with liver dysfunction ,37. Name1177) of eNOS via two distinct pathways—the PI3K-Akt and ERK1/2 pathway. Nevertheless, these two pathways seem to be activated with the same upstream regulator, one of the aforementioned adropin receptors—VEGFR2. In addition, adropin may also engage several NO-independent pathways, contributing to the regulation of endothelial function, through the VEGFR2-PI3K-Akt pathway, for instance, mobilization of bone marrow-derived endothelial progenitor cells. Finally, Lovren et al. demonstrated that adropin promoted indices of endothelial function, such as proliferation, migration, capillary-like tube formation, reduced permeability and apoptosis, as well as improved angiogenic potential [It has been well-established that the endothelium plays a central role in the maintenance of vascular homeostasis, and that impairment of endothelial function contributes to the development and progression of various pathologies, most notable with regard to the cardiovascular system . eNOS biotential .In the years following the above-noted study, multiple authors further explored the association between adropin and indices of ED in various clinical settings. Topuz et al. demonstrated that plasma adropin levels are reduced in type 2 diabetes mellitus (T2DM) patients with ED, as measured by brachial flow-mediated dilatation (FMD), whereas Oruc et al. demonstrated the same for patients with established metabolic syndrome using FMD as well ,41. Chan−/− mice.In a pivotal study by Sato et al., the authors provided the first evidence showing that adropin suppresses atherosclerosis independently of glucose and lipid metabolism, and blood pressure . In factThe clinical aspect of the role of adropin in ED has also been investigated in recent years. Multiple authors proposed that adropin might represent a novel biomarker for the presence of acute myocardial infarction (AMI), as well as predicting AMI onset in patients with coronary artery disease (CAD) ,47. Spec−/− mice [Respecting the effects of adropin on NO metabolism, it was examined whether adropin has a role in blood pressure regulation. Multiple authors reported higher adropin levels in hypertensive patients compared with normotensive controls ,50. On t−/− mice . However−/− mice .As NO is the main culprit of vascular stiffness, Fujie et al. sought to determine if adropin levels are associated with vascular stiffness . In the As reduced adropin plasma levels seem to be associated with various pathologies, and as they are most commonly associated with enhanced disease progression, it is merely a matter of time when adropin will be implemented in routine clinical practice. Specifically, since adropin correlates well with indices of ED, and ED being important yet poorly evaluated in clinical settings, this peptide represents a putative cardiovascular biomarker for the foreseeable future. Flow-mediated dilation is currently the most widely used method to study endothelial function. However, it is operator-dependent and can be influenced by physiological variations. Measurement of biomarkers such as adropin could overcome this issue. The biggest setback in adropin implementation is lack of understanding of the mechanisms in which it interferes with ED, thus leaving space for under recognition of confounding factors and lack of prospective large-scale studies. On the other hand, the main advantages are relatively low price, reproducibility, and according to the available data, substantial correlation with ED indices. Specifically, adropin has already shown favorable sensitivity/specificity profile for ED detection in patients with OSA.Nevertheless, each novel biomarker has to be assessed by its appropriateness to answer elementary questions in order to judge its clinical relevance: to which group of patients should the marker be applied to, at which point in time should the marker be measured, does the biomarker provide additional information beyond existing biomarkers, and finally, whether the biomarker has yielded auspicious features in a cost-effective analysis. Hence, in order to establish adropin as biomarker of ED in different pathologies, prospective large-scale studies with appropriately defined populations are required. Notably, the signaling mechanisms and receptors by which adropin acts on different cell types also need to be fully disclosed. Better understanding of these mechanisms will alleviate implementation of adropin for both diagnostic and therapeutic purposes.On the other hand, several authors conducted interventional studies, mostly on animals, with the intention to establish the therapeutic potential of adropin. As previously discussed, Sato et al. demonstrated that adropin exerts anti-atherosclerotic effects by suppressing monocyte–endothelial cell adhesion and smooth muscle cell proliferation. Considering the above-noted experiment, and adropin-induced upregulation of eNOS, it is plausible that adropin may be a novel target to limit a multitude of diseases in which ED is a culprit for poorer cardiovascular outcomes, exerting a vasoprotective role ,55. AdroIn the setting of DCM, adropin may be beneficial as well, since adropin treatment improved ex vivo cardiac function and resulted in better glucose tolerance and reduced insulin resistance in a model of mice with diet-induced obesity ,17. DCM Finally, discussing the therapeutic implications of adropin, there is an important limitation that we need to circumvent in order to implement adropin as a therapeutic agent. Adropin being a peptide hormone, its therapeutic delivery is rather challenging, as delivery of peptide hormones is fairly limited by their pharmacokinetics and potential for proteolytic degradation. Hence, better understanding of adropin signaling pathways is mandatory, as it will pave the way for the creation of small (more resistant) molecules mimicking the effects of adropin.In summary, adropin is a multifunctional highly conserved peptide implicated in various physiological and pathophysiological processes. Apart from its metabolic role, most importantly being the suppression of hepatic glucose production and improvement of insulin sensitivity, adropin seems to be an important gatekeeper of vascular health, and thus, an integral component of cardiometabolic diseases. Specifically, the vasoprotective role of adropin is achieved mainly by affecting endothelial NO synthesis. Furthermore, as plasma levels of adropin correlate with indices of ED in various pathologies and enhanced disease progression, adropin may serve as a useful biomarker in the upcoming future. Finally, the therapeutic potential of adropin in various pathologies has also been explored recently. Nevertheless, despite notable results in preliminary experiments, further well-designed studies are warranted in order to establish if adropin might be beneficial in this setting.


Early and timely fluid treatment or resuscitation are the basic measures for the active treatment of sepsis. Our aim is to further explore the relationship between fluid balance and prognosis in patients with sepsis on a daily basis for 5 days.Sepsis patients in eICU Collaborative Research Database were divided into the negative balance group (NB/−) and the positive balance group (PB/+) according to daily fluid balance. The primary outcome was in-hospital mortality. Survival differences between the groups were analyzed by using Cox regression. Then dose-response relationship between fluid balance and in-hospital mortality was studied using restricted cubic splines (RCSs). Furthermore, patients with fluid balance data for the previous three consecutive days were selected and divided into eight groups . Kaplan–Meier curves and Cox regression were used to show the survival difference between groups.Our study, which included 19,557 patients in a multicenter database, showed that positive fluid balances on days 1, 2, and 3 after sepsis diagnosis were associated with poor prognosis with the HRs of 1.29 , 1.13 , and 1.25 , respectively, while the fluid balance on days 4 and 5 had no effect on the primary outcome. Then RCSs showed an overall trend that the risk of in-hospital mortality on days 1, 2, and 3 increased with increasing fluid balance. For three consecutive days of fluid balance, we studied 9205 patients and Kaplan–Meier curves revealed survival differences among patients in the eight groups. The cox model demonstrated that compared with the “+/+/+” group, the “+/ −/−”, “−/ −/−”, “−/ −/+”, “−/+/+”, and “−/+/−” groups had a lower risk of in-hospital mortality, with HRs of 0.65 , 0.72 , 0.63 , 0.69 , and 0.63 , respectively.In patients with sepsis, positive fluid balance on days 1, 2, and 3 was associated with adverse outcomes. For patients with fluid balance for three consecutive days, the “+/−/−”, “−/ −/−”, “−/−/+”, “−/+/+”, and “−/+/−” groups were less likely to die in hospital than the “+/+/+” group. Sepsis is defined as life-threatening organ dysfunction caused by a host’s dysfunctional response to infection and is associated with a high incidence of morbidity and mortality worldwide . DespiteEarly and timely fluid treatment or resuscitation and necessary vasopressor use are the basic measures for the active treatment of sepsis. Among these methods, fluid therapy or resuscitation aims to correct the relative or absolute deficiency of blood volume through rapid fluid supplementation, to ensure normal cardiac output and organ blood perfusion, and to protect organ function . HoweverIn recent years, a growing body of evidence has shown that over-aggressive fluid resuscitation may have side effects. For example, a previous work  demonstrThe possibility of volume overload due to the improper infusion of large amounts of fluids is increasingly recognized as an independent risk factor for disability and death in critical illness . A prosphttps://eicu-crd.mit.edu/).The data analyzed in this study were collected from the eICU Collaborative Research Database (eICU-CRD), a public, multicenter ICU database that includes electronic medical records from 208 hospitals and data from more than 200,000 patients in 2014−2015 , 11. AllSepsis was diagnosed by using the latest criteria for sepsis 3 , which iTherefore, we extracted the information of infected patients with SOFA ≥ 2 from the eICU-CRD, among which 36,302 patients met the diagnostic criteria for sepsis 3. Exclusion criteria were as follows: patients < 18 years of age, patients who died within 24 h of admission to the ICU, and patients without fluid records. A total of 19,557 patients were included in this study.We use SQL (Structured Query Language) for data extraction. The patientunitstayid identifier of the patients with sepsis was used to extract the general information of the patient, including age, gender, weight, height, and ICU type; intervention measures: dialysis, ventilator, and vasopressor; comorbidity: stroke, congestive heart failure (CHF), hypertension, chronic obstructive pulmonary disease (COPD), renal failure, liver diseases, diabetes, and cancer; severity scores, namely, acute physiology and chronic health evaluation scoring system (APACHE) IV and Sequential Organ Failure Assessment (SOFA) scores; infection source; and the number of patients in and out 5 days after the diagnosis of sepsis. Day 1 was defined as 24 h after the diagnosis of sepsis. The daily fluid intake is calculated as the sum of all intravenous and oral fluids. The daily output is calculated as the sum of urine output, stool volume, emesis, blood loss, dialysis ultrafiltrate yield, drainage fluid volume, puncture fluid volume  etc. The invisible losses of liquids were not taken into account because they were difficult to estimate. The daily fluid balance was determined as the difference between the total intake and the total output and was divided into the negative balance group (NB/−) and the positive balance group (PB/+) on the basis of the difference.The primary outcome was in-hospital mortality, and the secondary outcome was duration of ventilator use.Categorical variables were described as frequency and percentage values, and differences between the two groups were determined by using the chi-square or Fisher exact test. The Shapiro–Wilk test was used to test whether continuous variables fit the normal distribution. Continuous variables that fit the normal distribution were described as mean and standard deviation values, whereas those that did not fit the normal distribution were described as median and quaternary range values.Cox regression was used to compare daily survival differences between the two groups. The hazard ratio (HR) and 95 % confidence interval (CI) were calculated by using multivariate Cox regression by controlling for the following confounders: age, gender, weight, height, unit type, dialysis, ventilator, vasopressor; comorbidities: stroke, CHF, hypertension, COPD, renal failure, liver disease, diabetes, and cancer; APACHE IV and SOFA scores; and infection source.After preliminary analysis, the fluid balances on days 1, 2, and 3 were found to have an influence on the in-hospital mortality. Therefore, we conducted further analysis. The RCSs was used to explore the dose-response relationship between fluid balance on and in-hospital mortality in sepsis patients on days 1, 2, and 3. Furthermore, patients with fluid balance data for the previous three consecutive days were selected and grouped. Kaplan–Meier curves were used for survival analysis, and Cox proportional hazard regression models were used to examine the effects of various factors on hospital mortality.All statistical analyses were conducted on R (version 4.0.3). A two-sided p-value of <0.05 was considered statistically significant.A total of 19,557 patients were included in this study. Table After controlling for potential confounders in Cox regression, the risk of in-hospital mortality was found to be statistically higher in the PB group than in the NB group on day 1 after the diagnosis of sepsis. The HRs (95CI%) of in-hospital mortality for the PB group were 1.29 . This result indicated that the risk for in-hospital mortality in the PB group was 1.29 higher than that in the NB group. The same trend was observed for the risks of in-hospital mortality on days 2 and 3 after sepsis diagnosis, which were 1.13 and 1.25 times higher in the PB group than in the NB group. No significant difference in the in-hospital mortalities between the two groups on days 4 and 5 were observed (Fig. The above results indicated that fluid balance on days 1–3 had an influence on the in-hospital mortality of patients after sepsis diagnosis. RCSs results showed a non-linear relationship between fluid balance volume and the risk of in-hospital mortality on days 1 and 2, while no such relationship was observed on day 3 (Fig. We selected a total of 9205 patients with fluid data for three consecutive days to further verify the effect of daily fluid balance on outcomes and divided them into the “+/+/+”, “+/+/−”, “+/ −/−”, “+/ −/+”, “−/ −/−”, “−/ −/+”, “−/+/+”, and “−/+/−” eight groups in accordance with their daily fluid balance. The Kaplan–Meier curve is shown in Fig. Linear regression showed that breathing machine use days differed between groups. For days 2–5, the “+” group had longer actual ventilator use days than the “−” group. For patients with fluid balance for three consecutive days, the “+/+/−”, “+/ −/−”, “−/ −/−”, “−/ −/+”, and “−/+/−” groups had fewer actual ventilator days than the “+/+/+” group (Table The pathological characteristics of sepsis are the reduction of effective circulating blood volume and the insufficient perfusion of tissues and organs in the body . Liquid Our study, which included 19,557 patients in a multicenter database, revealed that positive fluid balance on days 1, 2, and 3 after sepsis diagnosis was associated with poor prognosis, and the RCSs showed an overall trend of increasing the risk of in-hospital mortality with increasing fluid balance. We grouped patients with fluid balance records for the previous three consecutive days in accordance with daily fluid balance given that fluid administration is continuous and dynamic to further study the influence of fluid balance on patient mortality in the hospital. Compared with those in the “+/+/+” group, patients in the “+/ −/−”, “−/ −/−”, “−/ −/+”, “−/+/+”, and “−/+/−” groups were less likely to die in the hospital. The possible mechanism is that the vascular endothelial permeability of patients with sepsis is increased, and the overloaded fluid extravasates to cause tissue and organ edema, which is not conducive to the recovery of organ function and ultimately affects prognosis . PositivPatients receive excess fluid, resulting in fluid overload. In the middle and late stages of sepsis, the pathogenesis and course of sepsis in patients are complex, and fluid management may be affected by numerous factors, such as the patients’ basic physical conditions and complications, fluid types, and the target endpoint of fluid resuscitation. At the same time, a clear demarcation between the stages of shock does not exist. Therefore, in clinical practice, grasping the two aspects of adequate fluid resuscitation in the early stage and restricted fluid management in the late stage of fluid therapy remains difficult . Fluid tThe advantage of this study is that the eICU-CRD is a multicenter database, and its large sample size provides strong evidence for our study. In addition, we grouped the patients in accordance with their fluid balance on the first three consecutive days after diagnosis to further explore the mortality of patients in different combinations. However, this study has some limitations because it only investigated the relationship between positive fluid balance and mortality. Whether this relationship is a simple association or a causal relationship is not clear, and further confirmation with a large sample of prospective studies is needed.In patients with sepsis, positive fluid balance on days 1, 2, and 3 was associated with adverse outcomes. For patients with fluid balance for three consecutive days, the “+/−/−”, “−/ −/−”, “−/−/+”, “−/+/+”, and “−/+/−” groups were less likely to die in the hospital than the “+/+/+” group. In the treatment of sepsis, reasonable fluid therapy should be used, and continuous positive fluid balance is not recommended.


Trial registration: NCT NCT02142985. Registered 05 May 2014.2) measurement has been proposed to guide fluid therapy . Diagnostic performance of ΔScvO2 and ΔCVP after VE showed areas under ROC curves of 0.84  and 0.56 , respectively. The AUC of ΔScvO2 was significantly greater than that of ΔCVP . The best cut-off value found was 4%, allowing discrimination between responders and non-responders with a sensitivity of 78.7%, a specificity of 81.5% and a percentage of correct classification of 61.1%. It is important to mention that our results would work when ScvO2 is low and when there is no significant change in VO2 and haemoglobin. However, changes depend on the amount of fluid administered. Also, ScvO2 may not change if VO2 is dependent on DO2. We concluded that in patients with acute circulatory failure, ΔScvO2 has an adequate correlation with ΔCI and could be very helpful alternative tool when CO measurement or surrogates aren’t possible or not applicable. Further studies with larger populations are required to confirm these results on the role of ScvO2 monitoring in assessing fluid responsiveness instead of a cardiac output measurement during a fluid challenge.Intravenous fluid administration is a cornerstone of hemodynamic resuscitation. Its goal is to restore effective circulating blood volume and correct tissue perfusion as quickly as possible to avoid multi-organ failure . In extr therapy . The ratsponsive . We stud


Fluorescence imaging of tumours facilitates rapid intraoperative diagnosis. Thus far, a promising activatable fluorescence probe for hepatocellular carcinoma (HCC) has not been developed. Herein, the utility of the fluorescence imaging of HCC using a β-galactosidase -activatable fluorescence probe SPiDER-βGal was examined. β-Gal activity was measured in cryopreserved tissues from 68 patients. Live cell imaging of HCC cell lines and imaging of tumour-bearing model mice were performed using SPiDER-βGal. Furthermore, fluorescence imaging was performed in 27 freshly resected human HCC specimens. In cryopreserved samples, β-Gal activity was significantly higher in tumour tissues than in non-tumour tissues. Fluorescence was observed in HCC cell lines. In mouse models, tumours displayed stronger fluorescence than normal liver tissue. In freshly resected specimens, fluorescence intensity in the tumour was significantly higher than that in non-tumour liver specimens as early as 2 min after spraying. Receiver operating characteristic curves were generated to determine the diagnostic value of SPiDER-βGal 10 min after its spraying; an area under the curve of 0.864, sensitivity of 85.2%, and specificity of 74.1% were observed for SPiDER-βGal. SPiDER-βGal is useful for the rapid fluorescence imaging of HCC. Fluorescence imaging guided by SPiDER-βGal would help surgeons detect tumours rapidly and achieve complete liver resection. Hepatocellular carcinoma (HCC) accounts for most primary liver cancers. Surgical resection is one of the most effective treatments for HCC. However, > 70% of patients have tumour recurrence within 5 years after hepatectomy for HCC3. The presence of pathological cancer remnants after liver resection is a major recurrence risk4. Therefore, even minute amounts of residual cancerous tissues must be prevented in preserved livers. To achieve complete resection, it is important to identify the location of the tumour intraoperatively and confirm that no cancer is exposed at the resected surface of the liver. A modality helping surgeons detect HCC intraoperatively is therefore highly desirable.Liver cancer, the sixth most frequently diagnosed cancer worldwide and the fourth most common cause of cancer-related mortality5. Indocyanine green (ICG)12 and 5-aminolevulinic acid (5-ALA)15 are fluorescent molecules often used intraoperatively to facilitate the complete resection of HCC. Recently, activatable fluorescence probes, which are normally non-fluorescent but can be activated through cancer-specific enzymes, have been developed as novel fluorescent diagnostics that can specifically and rapidly image cancer after topical spraying. For example, a fluorescence probe targeting dipeptidylpeptidase IV has been used to detect oesophageal squamous cell carcinoma17 and adenocarcinoma of the oesophagogastric junction18. Similarly, a gamma-glutamyl transpeptidase (GGT)-activatable fluorescence probe can reportedly detect ovarian cancer19, breast cancer20, lung cancer21, liver cancer22, superficial head and neck squamous cell carcinoma23, and metastatic lymph nodes in colorectal cancer24. However, target enzymes and fluorescence probes that are sufficiently effective for use in HCC have not been determined. β-galactosidase -targeted fluorescence probes can help visualise ovarian cancer cells, and the small peritoneal metastases from ovarian cancer in mice26. Recently, we reported that β-Gal is a target enzyme for detecting the peritoneal metastasis in gastric cancer27. However, the fluorescence imaging of HCC using β-Gal-targeted fluorescence probes has not been reported.Fluorescence-guided surgery has been developed as a safe and reliable surgical method28, for the intraoperative rapid fluorescence imaging of HCC.This study focused on β-Gal as a candidate enzyme for the fluorescence imaging of HCC and aimed to examine the feasibility of using the SPiDER-βGal, β-Gal-targeted fluorescence probe22, and found that GGT activity was significantly lower in tumour tissues than in non-tumour tissues  and hepatitis C virus (HCV) infections was significantly higher than that in individuals with HBV and HCV infections Table . HoweverTo investigate the use of SPiDER-βGal, we treated HCC cell lines  and HUVECs with SPiDER-βGal (1 μM). The intracellular fluorescence signal in HCC cell lines increased after this treatment. However, intracellular fluorescence was low in HUVECs after 60 min of SPiDER-βGal treatment Fig. . QuantifFurthermore, we assessed the potential application of SPiDER-βGal for fluorescence imaging of tumours in four different tumour-bearing mouse models. We successfully established these four tumour mouse models using Hep-G2, HuH-7, PLC/PRF5, and Li-7 cells. After spraying the cut surface of tumours with 50 μM SPiDER-βGal, fluorescent signals were compared with those in normal livers from Hep-G2, HuH-7, PLC/PRF5, and Li-7 tumour-bearing mice Fig. a. In HuHFreshly resected human HCC samples, obtained from 27 consecutive patients from May 2019 to March 2020, were sprayed with SPiDER-βGal (50 μM) and imaged every 2 min up to 30 min. The patient clinicopathological characteristics are indicated in Supplementary Table 11 and 5-ALA15 as fluorescence imaging agents in liver cancers. However, both ICG and 5-ALA requires preoperative administration for cancer imaging. On the other hand, activatable fluorescence probes do not require preoperative preparation and can detect cancers simply through topical spraying, whenever surgeons need to identify cancer. Our current study suggested that SPiDER-βGal is useful for cancer diagnosis in freshly resected human HCC specimens.Fluorescence-guided surgery is potentially safer and more reliable for liver surgery than conventional surgery. Several previous studies have reported the use of ICG29. Therefore, haemoglobin in the RBCs of Hep-G2-derived tumours may have influenced the difference observed in the live-cell imaging and mouse model imaging results.In live-cell imaging using SPiDER-βGal, Hep-G2 cells exhibited fluorescence; however, the Hep-G2-derived subcutaneous tumours in mice, which possessed more RBCs than the other cell line-derived subcutaneous tumours, showed a slight increase in fluorescence intensity after application of SPiDER-βGal. Furthermore, haemoglobin reportedly quenches fluorescenceFluorescence imaging of freshly resected human HCC specimens with SPiDER-βGal is useful; however, the difference in the increase in fluorescence intensity between the tumour and normal liver in a freshly resected human sample during imaging using SPiDER-βGal was greater than the difference in the degree of β-Gal activity of the lysate between the tumour and normal liver. Tumours that showed a slight increase in fluorescence intensity exhibited a reddish colour in white light image  staining has reported that some HCC liver tumour cells express β-Gal32; however, details of β-Gal activity in HCC remains unknown. Our results suggested that individuals without HBV and HCV infections have high β-Gal activity in tumour tissues. HCV core protein was reported to inhibit HCC cell replicative senescence33. Further, β-Gal is known to be a senescence marker34. So, β-Gal activity may be influenced by a hepatitis virus infection. In order to improve the diagnostic performance of the β-Gal-targeted fluorescence probe, further study of the association between the hepatitis virus and β-Gal are necessary in the future.Tumour β-Gal activity has been reported to be especially high in breast cancer, colon cancerThus, this study indicates that SPiDER-βGal is useful for the rapid fluorescence imaging of human HCC. SPiDER-βGal might be applicable for intraoperative diagnosis. In particular, fluorescence-guided surgery using SPiDER-βGal is considered useful for determining whether the tumour is exposed at the resected liver surface. Fluorescence-guided surgery with SPiDER-βGal might facilitate complete liver resection and reduce HCC recurrence.Tissue samples from the site of tumour and non-tumour liver parenchyma were harvested and cryopreserved from patients undergoing a curative liver resection at the University Hospital of Kyoto Prefectural University of Medicine (KPUM). A consort flow chart is provided in Fig. [™] Supplements and Growth Factors (Lonza) in collagen I-coated dishes . All cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.Four HCC cell lines and human umbilical vein endothelial cells (HUVECs) were used in this study: Hep-G2, HuH-7, and Li-7 cell lines ; PLC/PRF/5 cell line ; HUVECs . HuH-7, Hep-G2, and PLC/PRF/5 cells were cultured in DMEM . Li-7 cells were cultured in RPMI1640  Both medium contained 10% heat-inactivated foetal bovine serum , penicillin (100 U/mL) and streptomycin (100 μg/mL) . HUVECs were cultured in Endothelial Cell Basal Medium-2  and Endothelial Cell Growth Medium-2 SingleQuotsg for 10 min at 4 °C) to pellet cellular debris. The supernatant was then harvested and assessed for protein concentration using a BCA protein assay kit , in accordance with the manufacturer’s instructions. Subsequently, the supernatant was diluted to 1 mg/mL with D-PBS .Cryopreserved tissues were suspended in CelLytic M  and finely chopped using scissors. Thereafter, the chopped tissues were homogenised using an ultrasonic homogeniser on ice. The lysed tissues were centrifuged . Then, 50 μL of Na2CO3 (0.2 M in H2O) was added to all wells to terminate the reaction and the fluorescence intensity (Ex/Em: 390/460 nm) was measured using a micro plate reader . For the β-Gal activity assay at pH 7.4, the plate was incubated for 30 min . Subsequently, the fluorescence intensity was measured. The intensity of each sample was normalised against that of the reference standard.β-Gal activity was evaluated in 96-well black plates  using acetate buffer (pH 5.0)  or D-PBS (pH 7.4) and a FluoReporter lacZ/Galactosidase Quantitation Kit , in accordance with the manufacturer’s instructions. First, tissue lysate samples  were added to triplicate wells. Thereafter, 3-carboxyumbelliferyl β-D-galactopyranoside (1.1 mM) prepared in acetate buffer  or D-PBS (pH 7.4) was added to the wells. Acetate buffer (10 μL/well) and 7-hydroxycoumarin-3-carboxylic acid (0.1 mM) diluted with acetate buffer (100 μL/well) or D-PBS (pH 7.4) (10 μL/well) and 7-hydroxycoumarin-3-carboxylic acid (0.1 mM) diluted with D-PBS (100 μL/well) were added to triplicate wells as a reference standard. For the β-Gal activity assay at pH 5.0, the plates were incubated for 30 min . Tissue lysates  and fluorescence probes  were added to each well. HMRG was used as a reference standard, and gGlu-HMRG was used as a fluorescence probe. Fluorescence intensity was measured using an EnVision multilabel plate reader  every minute for 120 min . The results of the gGlu-HMRG assay were normalised to those of the HMRG assay, which was concurrently performed. GGT activity was determined using the following formula:4 cells/dish) were seeded in the centre of 35-mm glass-bottom dishes  and incubated in an atmosphere containing 5% CO2 at 37 °C for 1–2 d. Thereafter, the cells were washed twice with Hanks’ Balanced Salt solution . Next, SPiDER-βGal (1 μM) was added to the dishes, and the cells were incubated in 5% CO2 at 37 °C for 60 min. Fluorescent images were obtained using a Keyence BZ-X800 with the TRITC filter . Bright-field images were captured simultaneously. As a control, we added an identical volume of HBSS to the dishes of cultured cells instead of SPiDER-βGal. The fluorescence intensities of 10 randomly selected cells were analysed using Image J version 1.52a (NIH).Cells  were mixed with an equal amount of Matrigel  on ice. Under general anaesthesia, the mixed suspension (100 μL) was then subcutaneously injected into the flanks of each mouse. After ≥ 4 weeks, tumour-bearing mice were euthanised using Isoflurane . Subcutaneous tumours and livers were dissected and divided into two using scissors. A solution of SPiDER-βGal (50 μM) in HBSS was then sprayed onto the cut surface of each tumour or the surface of normal liver. Sequence of fluorescent images were captured every 2 min for 30 min using IVIS Lumina Series III (Ex/ Em: 520/570 nm). Regions of interest (ROIs) were drawn for both the tumour and normal liver tissues, and the average radiant efficiency was determined as fluorescence intensity using Living Image version 4.4.All animal experiments were performed in compliance with both the ARRIVE guidelines and the institutional guidelines of KPUM, and approved by the animal experimental committee of KPUM . Five-week-old female BALB/c nu/nu mice  were purchased from SHIMIZU Laboratory Supplies, Japan. The mice were housed in plastic cages with stainless-steel grid tops in an air-conditioned environment with a 12-h light–dark cycle and were fed regular food and water ad Resected subcutaneous tumours from tumour-bearing mice were fixed with 10% neutral buffered formalin then embedded in paraffin. The paraffin blocks were sliced to a thickness of 5 μm, after which the paraffin-embedded sections were deparaffinised and stained with Mayer’s haematoxylin solution (Wako) and eosin Y (Wako) for histopathological analysis.35.Freshly resected human specimens were obtained from patients preoperatively diagnosed with HCC—through radiological examination—who received curative liver resection at the University Hospital of KPUM. Written informed consent was preoperatively acquired from all patients. The Institutional Review Board of KPUM examined and approved the research procedures  in accordance with the tenets of the Declaration of Helsinki. Cases of cholangiocellular carcinoma without HCC components, as determined pathologically, were excluded. In total, 27 freshly resected human HCC specimens were examined prospectively from May 2019 to March 2020. Patient clinicopathological characteristics were described based on the General Rules for the Clinical and Pathological Study of Primary Liver Cancer, Edition 6, Revised VersionFluorescence imaging of human specimens was performed within 1 h after liver resection. A solution of SPiDER-βGal (50 μM) prepared in HBSS was then sprayed onto the resected surface of the liver tissue samples. Sequences of fluorescent images were captured every 2 min for 30 min using IVIS Lumina Series III (Ex/Em: 520/570 nm). ROIs were drawn for both the tumour and normal liver, and the average radiant efficiency as a fluorescence intensity was determined using Living Image version 4.4.t-test was used to compare β-Gal activity in cryopreserved human HCC samples at pH 5.0 and pH 7.4, and GGT activity in cryopreserved human HCC samples. A two-tailed Mann–Whitney U-test was used to compare the fluorescence intensity of live cells, fluorescence intensity of images from tumour-bearing mouse models, and clinicopathological characteristics of human HCC tissue samples that were either cryopreserved or freshly resected. A two-tailed Wilcoxon t-test was used to compare the fluorescence intensity of freshly resected HCC specimens. ROC curves were constructed using JMP13 . ROC curves and Youden index were used to determine the optimal cut-off value for β-Gal activity in the cryopreserved human HCC samples and for assessing the increase in fluorescence intensity in freshly resected HCC specimens. The sensitivity, specificity, and area under the curve (AUC) were determined through ROC analysis. Results with p-values < 0.05 were considered significant. Statistical analysis was performed using the yStat 2013 software.A two-tailed paired Supplementary Information.


In emitters with the orthogonaldonor and acceptor fragments, such spin-forbidden 1CT ↔ 3CT transitions are activated by molecular vibrations. Namely,the change of dihedral angle between the donor and the acceptor affordsreasonable spin–orbit coupling, which together with a smallenergy gap and reorganization energy enable 1CT ↔ 3CT transition rates reaching 1 × 107 s–1. Evidence of direct 1CT ↔ 3CT spin-flip and negligible role of a second triplet state,widely believed as a key parameter in the design of (r)ISC materials,change significantly the current understanding of TADF mechanism.In authors’ opinion, photophysics, and molecular design principlesof TADF emitters should be revised considering the importance of vibrationallyenhanced 1CT ↔ 3CT transitions.Thermally activateddelayed fluorescence (TADF) has recently becomean extensively investigated phenomenon due to its high potential forapplication in organic optoelectronics. Currently, there is stilllack of a model describing correctly basic photophysical parametersof organic TADF emitters. This article presents such a photophysicalmodel describing the rates of intersystem crossing (ISC), reverseISC (rISC), and radiative deactivation in various media and emphasizingkey importance of molecular vibrations on the example of a popularTADF dye 9,10-dihydro-9,9-dimethyl-10-phenyl)-acridine(DMAC-TRZ). The presented experimental and theoretical investigationsprove that ISC and rISC can occur efficiently between the singletand triplet states of the same charge-transfer nature ( Over the last decade,fast development of all-organic optoelectronicswith an increasing number of potential applications provided a plethoraof experimental solutions on the (sub)molecular level. In one of themost promising applications of all-organic light-emitting diodes (OLEDs),these solutions are mainly focused on the problem of triplet harvesting.Due to spin statistics, recombination of charges in organic semiconductorsleads to formation of singlet (25%) and triplet (75%) excitons. Inheavy-atom free molecular systems, triplet excitons usually cannotensure fast rates of light emission and should be converted to thesinglet ones for high electroluminescence efficiency.1 or by common fluorescent emitters dispersed inTADF emitters in the role of hosts, the so-called “hyperfluorescence”approach.2 Numerous photo- and electroluminescentinvestigations revealed strong dependence of internal and externalquantum efficiencies on the rISC rate. The importance of materialswith fast rISC for further development of optoelectronics cannot bethus overestimated.Currently,probably the best solution for triplet harvesting isthe use of materials with fast reverse intersystem crossing (rISC).This can be realized either by emitters exhibiting thermally activateddelayed fluorescence (TADF) dispersed in common OLED hosts1CT and triplet 3CT electronic states.Efficient separation of the donor and acceptor provides a negligibleoverlap of highest occupied molecular orbital (HOMO) and lowest unoccupiedmolecular orbital (LUMO), which decreases the exchange and energygap (ΔE1CT–3CT) between the 1CT and 3CT states. Alltheoretical models developed before to describe the bases of TADFphotophysics follow the selection rule stating that spin-flip betweenthe 1CT and 3CT states is forbidden due to theiridentical nature. In fact, state-of-the-art methods of quantum chemistrypredict a zero spin–orbit coupling constant (SOCME) for the 1CT and 3CT interaction. For this reason, TADF efficiencyis believed to be strongly dependent on the presence of at least oneadditional triplet state with close energy to the 1CT and 3CT ones. This can be either a locally excited 3LE state localized on a donor or an acceptor3 or another CT state formed via electronic transition on differentmolecular orbitals.4 According to this,rISC is mediated by this third state because the transition betweenit and the 1CT state is more allowed due to their differentnature. The presence of such a third state is believed as anotherkey requirement in the design of TADF emitters.Up to the moment, there has been no generaltheoretical model whichwould correctly describe the basic photophysical parameters of TADFmaterials such as rates of intersystem crossing (ISC), rISC, and radiativedeactivation in various media. Obviously, this impedes correct understandingof the mechanism and design principles of efficient TADF materials.In this article, we present such a model and on the example of oneof the most popular TADF emitters explain how high rISC and ISC ratesare achieved under various conditions. To date, all organic emitterswith superior rISC rates are built of spatially separated strong electronicdonor and acceptor molecular fragments, which is the key principleof design strategy of TADF emitters. Such a structure enables formationof charge-transfer (CT) states, which represent the lowest excitedsinglet 3LE state has been used to explainthe majority of TADF examples including a triazine derivative 9,10-dihydro-9,9-dimethyl-10-phenyl)-acridine(DMAC-TRZ) investigated here. It should be emphasized that such athree-state model predicts maximum rISC rate when 3LE and 3CT/1CT states are energetically close. In termsof spectroscopic parameters measured experimentally, this means thatthe energy gap between 1CT and 3LE states (ΔE1CT–3LE) shouldbe zero. More advanced variations of the three-state model specifyan additional condition for maximal rISC: vibronic coupling between 3LE and 3CT states.5 Similarcriterion is found in another theoretical model, which assumes thatthe T1 state of various s-triazine andbenzonitrile emitters is not of pure CT-nature but contains variousportions of LE nature.6 Such a model thussuggests different nature of S1 and T1 states,which provides relatively high SOCME values and in such a way explainsefficient rISC. The extent of LE contribution can be substantial ina non-polar medium, when CT energy is close to the triplet energyof a separate acceptor or donor, but it should decrease when CT isstabilized in high-polarity media. As follows from all these models,when the lowest CT states are stabilized and the ΔE1CT–3LE energy gap increasesby module, the rISC rate should decrease sharply. What is more, thesame conclusion should be true for the forward ISC transition becauseit should also strongly depend on ΔE1CT–3LE. Here, we provide evidencethat such conclusions on the dependence of ISC and rISC rates on theΔE1CT–3LE value are not supported by the experiment.Among the above-describedcases, the one relying on the energeticallyclose donor or acceptor S,S-dioxide emitters even in liquid solutions.7 In this case, in spite of low ΔE1CT–3LE value,the restriction of some undetermined molecular vibrations by methylgroups was concluded to be extremely important for rISC: the morethe vibrational freedom was restricted, the less TADF efficiency wasobserved. In the case of less flexible derivatives with more bulkyisopropyl or tert-butyl groups, TADF was almost absentand the T1 state deactivated mainly via room-temperaturephosphorescence. Next, it was found that the explanation of the radiativerate of TADF emitters with orthogonal donor and acceptor fragmentsis not possible within “frozen” optimized geometry becauseof zero oscillator strength predicted by calculations.8 It was proved that molecular vibrations should be takeninto account for adequate prediction of oscillator strength and electronictransition moment. In another recent investigation, the behavior ofDMAC-TRZ in solid solutions was explained by the inhomogeneity ofits geometry caused by rotational isomerism and distribution of CTstates.9 Another recent theoretical modelassumed that both spin–orbit coupling  and the so-called direct SOC (T1 → S1) play important roles in rISC.10 However, the direct T1 → S1 transition was predicted to be efficient when the natureof T1 and S1 states is different: the presentedcalculations predicted 92% CT nature for the S1 state and95% LE nature for the T1 state. The proposed model canthus also be valid only in the case of the proximity of 1CT and 3LE states. Despite the fact that the above-mentionedinvestigations were still explained by the three-state model, theseexperimental results question the primary importance of minimal ΔE1CT–3LE value fora maximum rISC rate at T > 0 K where moleculesexistin various excited vibronic states.Some previous articleshave already pointed at some discrepanciesbetween the three-state model and the experimental behavior of TADFemitters. Namely, some of the investigations evidence the key roleof molecular vibrations in TADF photophysics. For example, introductionof methyl groups at positions 1 and/or 9 of phenothiazine donor decreasesdrastically TADF rate and efficiency of phenothiazine-dibenzothiophene-11 Fast rISC in such emitters was explained bythe thermally activated rotation along the bond between the metalatom and the donor fragment, which decreased the ΔE1CT–3CT value and facilitateddirect T1 → S1 transition. Together withthe reasonable SOC values12 due to heavy-atomeffect, this resulted in high TADF rate constant (ca. 3 × 106 s–1). Further investigations of similarcompounds revealed that the energetic closeness of 3LEstate itself does not reduce the activation energy of rISC but resultsin the undesired decrease of emissive rate of the S1–S0 transition.13The importance of molecularvibrations for efficient TADF was alsosuggested for the carbene–heavy-metal complexes.1CT and 3LE statesshould favor the interaction between them. However, different electronicstructures lead to the differences in geometry. In terms of Marcussemiclassical electron-transfer theory,6 this means the increase of reorganization energy of 1CT ↔ 3LE transitions (λ1CT↔3LE) and should not increase but rather decrease theirrates. The investigations presented here prove that minimization ofreorganization energy is as much important as small energy gap betweensinglet and triplet state (ΔEST)value for achieving high rates of ISC and rISC. Substantial differencesin the reorganization energies play the decisive role in favor of 3CT ↔ 1CT transitions but not the 1CT ↔ 3LE ones.It shouldalso be noticed that according to the three-state model,the differences between the 3LE state for spin-fliptransitions, both ΔE1CT–3CT and ΔE1CT–3LE valueswere scanned experimentally. One of the most simple and basic butreliable spectroscopic experiments was conducted—solvatochromicmeasurements. The use of media of different polarities provides informationon the effect of alignment of energy levels on all basic photophysicalparameters, which is especially valuable for understanding TADF photophysics.DMAC-TRZ emitter was selected, which together with its various derivativesexhibits one of the best photo- and electroluminescent characteristicsextensively studied lately.15 Due to the high transitiondipole moment of the 1CT and 3CT states, theirenergies were effectively decreased with increasing medium polarity.Localized transitions are much less sensitive to the polarity of medium;therefore, the 3LE value is concerned as a constant. Thus,manipulation of solvent polarity, in a common notion meaning overallsolvation capability, allowed us to tune ΔE1CT–3LE from positive  to negative  values. Importantly, evidence was found that theenergy gap between 1CT and 3CT states also changeswith polarity. In spite of experimental simplicity of solvatochromicexperiment, it remains a challenging task for calculations using themethods of time-dependent density functional theory (TDDFT). For thisreason, the results of calculations were treated here with specialattention and analyzed only in the case of good correlation with experimentaldata.In this research, to checkthe widely accepted importance of 16 and purified by sublimation in vacuum. Solventsfor photophysicalmeasurements were of spectroscopic grade or higher. All the photophysicalmeasurements were conducted in the argon atmosphere.DMAC-TRZ was synthesized as reported previously17 Time-dependentemission measurements at room temperature were obtained using a FluoTime300 fluorescence lifetime spectrometer equipped with a LDH-P-C-375laser head. The parameters of photophysical processes were calculatedusing equations described in the literature;19 for details, see the Supporting Information.Steady-statephotoluminescence spectra were obtained with a PTIQuantaMaster 40 spectrofluorometer. All the emission spectra werecorrected on the photodetector sensitivity. Photoluminescence quantumyields (PLQY) were obtained using 9,10-diphenylanthracene in cyclohexane(93%) as a reference.0), excited singlet (S1), and triplet  electronic states at DFT/TDDFT levelof theory20 using the Gaussian 16 programpackage.21 The B3LYP22 hybrid functional was used with the cc-pVDZ basis set.Nature of states (CT or LE) was established on the basis of analysisof molecular orbitals involved in each transition, for details, seethe Supporting Information, Scheme S1.The energies of 1CT–S0, 1CT–3CT, and 1CT–3LE transitions wereobtained by single-point calculations using the optimized geometryof 1CT state (S1); the respective values forthe 3CT–S0, 3CT–1CT and 3LE–S0, 3LE–1CT transitions were obtained by single-point calculationsusing the optimized geometries of 3CT (T1) and 3LE (T2), respectively. Computational ΔE1CT–3CT and ΔE1CT–3LE valuesrepresent differences between minimal energies of respective states.The electronic energies and transition energies of rotational isomersin 1CT, 3CT, and 3LE states werecalculated by single-point calculations via dihedral angle scan usingthe geometries of the respective states. The SOC constants were calculatedwithin the ORCA program package,23 usingZORA relativistic contraction of the TZVP basis set.The unconstrained geometryoptimizations of DMAC-TRZ were performed for the ground  reorganization energies for therespective transition, ΔEST is anenergy gap between 1CT and the respective triplet state, kB is the Boltzmann constant, h̵ is thereduced Planck’s constant, and T is the temperature(298.15 K). For detailed procedures and examples of calculations,see the Supporting Information.For eachrotational isomer, the rate constants of ISC and rISC via 1CT state with the increase of medium polarity.Taking into account the 440 nm onset of 3LE phosphorescencemeasured in frozen methylcyclohexane solution,16 the ΔE1CT–3LE values in these experiments varied from +0.1 eV inhexane to −0.4 eV in acetone. In further discussion, to analyzethe medium effect and avoid fluctuations of composition of solventmixtures during the removal of oxygen, onset of fluorescence spectrum(E1CT) is used as an internalpolarity parameter.Supporting Information). The rate constantof radiative deactivation (kr) exhibitedstrong exponential dependence on E1CT , in a low-polarity medium, the rate constant ofnonradiative deactivation (knr) of 1CT state is minimal, when ΔE1CT–3LE is close to zero. One can thussuggest that the energetic closeness of 1CT and 3LE states is important for high PLQY. In more polar solvents, lower 1CT level corresponds to faster nonradiative deactivation.Importantly, the increase of knr is thekey factor decreasing PLQY in polar media but not the changes in ISCor rISC rates. In polar media, the main nonradiative deactivationchannel is most likely internal conversion, the rate constant of whichis proportional to 1/ΔE1–S0S2.25 In fact, knr shows good correlation with 1/ΔE1–S0S2 in polar media   decreases sharply. Despite that ΔE1CT–3LE becomesnegative increasing the driving force of 3LE → 1CT transition, decreased population of the 3LEstate should result in krISC decrease,which is not the case.To present further evidence of Δn(krISC) 2C. Inte changes 3: 3CT be1CT↔3LE and driving force ΔE1CT–3LE for the transitionsbetween the 1CT and 3LE states of differentgeometries. Within the Marcus theory, the maximum rate of 3LE → 1CT transition is not predicted for the sameenergy of the 1CT and 3LE states because ofthe additional reorganization energy involved. If rISC occurs viathe 3LE → 1CT transition, the dependenceof krISC on E1CT should contain two regions. First is “normalregion”, where −ΔE1CT–3LE < λ1CT↔3LE .Clearly, these regions are not observed in the experimental krISC on E1CT dependence.kr  between the donor andacceptor is changing  as compared to 1CT (13.20 cm–1). Consequently,the energies .For this reason, mean SOCME values of 0.043 and 0.051 cm–1 are obtained for the 1CT → 3CT and 3CT → 1CT transitions, respectively. Theincrease of SOC under deviation from orthogonality is explained bythe increasing overlap of molecular orbitals involved in CT transitions,namely, increased contribution of the phenyl-s-triazinefragment in HOMO . Within the Marcus theory, internal and external (solvent) reorganization energies are distinguished.For the 3CT → 1CT transition, calculationspredict very low structural reorganization energy <0.5 meV, whichis independent of θ within 90–65°. Due to a verysimilar geometry and electronic parameters of these two states, solventreorganization energy (λsolv) should also be low.One can expect that similar to ΔE1CT–3CT, the λsolv value should increase together with the growing difference betweenthe electronic parameters of the 3CT and 1CTstates. In further calculations, we thus assumed that the sum of reorganizationenergies for 3CT ↔ 1CT transitions (λ1CT–3CT) is equal to ΔE1CT–3CT.Prior to the theoretical prediction of rISC rate using the Marcus–Hushequation see , the eff3CT → 1CT transition forvarious rotational isomers using the Marcus–Hush equation andcalculated ΔE1CT–3CT and SOCME values  gives a k3CT→1CT value of 2.26 × 106 s–1. This value matches perfectly the experimental krISC value of 2.1 × 106 s–1 in o-dichlorobenzene. The predicted S1–S0 transition maximum of 611 nm is also in goodcorrelation with the experimental emission maximum in o-dichlorobenzene at 570 nm.The rISC rate constant calculated as a statistical sum of the rateconstants of 3CT → 1CT transition can solely afford highrISC rates due to molecular vibrations. High rate of 3CT→ 1CT transition is a combination of couple of factors.First is vibrationally activated SOC between the same CT states ofdifferent multiplicities. Even though it is 10–20 times smallerthan the SOC between the 3LE and 1CT states,the small ΔE1CT–3CT energy gap together with almost identical geometryand thus comparably small reorganization energy enable fast 3CT → 1CT rate. As discussed further, the reorganizationenergy of 3LE → 1CT transition is muchlarger, which is a decisive factor in favor of 3CT → 1CT transition.Such a remarkable match of experimentaland computational dataleads to two main conclusions. First, the B3LYP calculations of D–A-typeemitters correlate very well with the experiment in the medium ofrelatively high polarity. The most important conclusion is that the krISC in polar media (E1CT ≤2.67 eV) was done. Under such conditions, according to the Boltzmanndistribution, the population of 3LE state is below 0.2%and thus 3LE → 1CT transition cannotcontribute considerably to rISC. The 3CT → 1CT transition is thus the only pathway of rISC and its parameterscan be estimated. The ΔE1CT–3CT values corresponding to experimental krISC according to the Marcus–Hush equation arepresented in Table S1 (Supporting Information). The thus-obtained ΔE1CT–3CT values decrease with the rise of medium polarity,which supports perfectly the conclusions drawn above based on theexperimental kr values .The E1CT ≥ 2.72 eV, the molar fractions of speciesin the 3LE state should be taken into account . To calculate the rateof 3LE → 1CT transition, the experimentalΔE1CT–3LE and computationally predicted structural reorganization energies for various rotational isomers were used . The thus-obtained k3LE→1CT (λsolv = 0.3 eV) multiplied by the molar fraction of moleculesin the 3LE state at 298 K (χ3LE) are plotted in solv, seethe Supporting Information). Accordingto this, the 3LE → 1CT transition shouldhave the key contribution to rISC in the region of low polarity with E1CT close to 2.8 eV and ΔE1CT–3LE close tozero. In this region, the three-state model predicts maximum rISCrate, which is well described by the dependence of calculated k3LE→1CT on E1CT. This however completely contradictsour experimental data: krISC do not reachany maximum at ΔE1CT–3LE ≈ 0. In contrast, the experimental dependenceof krISC on E1CT is almost perfectly described by the rates of 3CT → 1CT transition, but by neither the 3LE → 1CT one nor the sum of rates of thesetwo transitions even in the low-polarity region , an ISC rate constant (kISC) value of 3.4 × 107 s–1 is the highest, but it decreases sharply to 1.5 × 107 s–1 in the benzene–hexane  mixture,where the E1CT value is 2.84eV. A further decrease of E1CT causes gradual increase of kISC up to3.1 × 107 s–1 in acetone. Accordingto the alignment of excited states, the decrease of E1CT in polar media makes the 1CT→ 3LE transition endothermic  is 4.2 times underestimated as compared to the experimental valuein o-dichlorobenzene . It should be noticed that the discussedapproach is a simplified model based on the first-mode vibrations.Even though these vibrations have the key influence on the electronicproperties of the CT states due to the change of dihedral angle θbetween donor and acceptor planes, some other vibrations of higherenergies also reduce the separation of HOMO and LUMO -9,9-dimethylthioxanthene-S,S-dioxide (DDMA-TXO2) exhibited fasterrISC in the DPEPO host than in Zeonex films,27 OLED devices containing tBuCzDCNPy emitter showed more than 3 timeshigher EQE when the DPEPO host was used instead of less polar mCP,28 as well as TTAZ and TXAZ emitters dispersedin the DPEPO host instead of the less polar medium, the mixture ofmCP and TSPO1 (1:1) hosts.29The analysis ofexperimental reports on other emitters proves that within similarmolecular systems, the highest rISC and/or external quantum efficiency(EQE) is achieved when the CT states are most stabilized and thus,as concluded above, ΔE1CT of a selected emitter usually does not exceed0.15 eV, which is too small to study a case with large ΔE1CT–3LE. Anotherimportant remark: in TADF emitters bearing conjugated fragments suchas phenothiazine, phenoxazine, phenyl-s-triazine,phenylpyrimidine, phenazine, benzophenone, naphthalimide, and so forth,as well as some carbazole derivatives and similar aromatic heterocycles,the 3LE energies are relatively low and, in non-polar medium,represent the T1 states. Numerous TADF emitters can serveas examples: PTZ-DBTO2,30 DPTZ-DPTO2,31 and their derivatives based on which the three-statemodel was mainly developed, numerous s-triazine derivatives,32 DBT-BZ-DMAC,33 NAI,34 various indoloindole derivatives,35 POZ-DBPHZ,36 andso on. When polarity increases, the CT states of such emitters aregetting energetically closer to 3LE, but simultaneously,the energy difference between 1CT and 3CT statesalso decreases, which most likely plays the key role in TADF.Almostall previous theoretical models were developed on the basis of experimentalphotophysical data in the media of low polarity such as OLED hosts or polymer matrixes such as Zeonex, polystyrene,and PMMA. Within such media, the variation of 16 bearing strong s-triazine acceptor shows higher rISC rate (7 × 105 s–1) as compared to its weaker 2-pyrimidine(no TADF) and 4-pyrimidine analogues (6 × 104 s–1).37 In the DPEPO host,despite a larger absolute ΔE1CT–3LE value, CT states of DMAC-TRZ are morestabilized and afford EQEmax of 25%, in contrast to thepyrimidine analogues not exceeding 8% and 12%, respectively. Similarly,the change of spiroacridine donor to a weaker phenazasiline one incombination with the same aryl-1,3,5-triazine acceptors led to destabilizationof CT states, slower TADF, and lower EQE.29The second way to change the CT-state energies is chemical modification.According to the literature reports, within derivatives bearing similardonor and acceptor fragments, better TADF parameters are observedin the emitters with higher donor/acceptor strength. For example,in toluene, DMAC-TRZE1CT–3CT value of < 2 meV and a large ΔE1CT–3LE value of 0.2 eV servesas an excellent proof for the key importance of energetic closenessof 3CT and 1CT . Taking3CT ↔ 1CT transformationis the vibrationally enhancedSOC with an average value of 0.05 cm–1. In one ofthe pioneering works, the phenomenon of hyperfine coupling betweenthe 1CT and 3CT states was suggested as a mainreason explaining fast population of T1 state in TADF emittersin time-resoled EPR experiment.39 Accordingto this model, SOCME due to hyperfine interaction can however reacha maximum of 0.2 cm–1, when ΔE1CT–3CT is below 0.051–0.025meV (0.4–0.2 cm–1). This can be valid forradical pairs; however, in almost all known molecular TADF emitters,the ΔE1CT–3CT value is hundred times higher. A conclusion on negligibleimpact of hyperfine coupling on the TADF photophysics was also madepreviously on the basis of theoretical modeling.5As concluded above, one of thefactors of efficient 40 It should be mentioned thatdonor and acceptor in these emitters are not completely orthogonal,which provides HOMO/LUMO overlap, differences in the S1 and T1 states’ nature, and thus non-zero SOC withlarge ΔEST already in the optimalgeometry.41 However, the ESP results consistentwith the vibronically assisted ISC model indicate that molecular vibrationsplay the key role in facilitating direct ISC and rISC via the S1 ↔ T1 in such compounds. Therefore, themodel presented here should be valid not only for D–A emitterswith orthogonal structure and well-separated frontier molecular orbitalsbut also for other kinds of CT TADF emitters with appropriate ΔEST values.Finally, the vibronically assisted ISC model wasapplied for the2CzPN and 4CzIPN emitters to explain the electron spin resonance (ESP)experiments.1CT and 3LE states lead to large internaland external reorganization energies for the 1CT ↔ 3LE transitions. To summarize the key numerical data for DMAC-TRZ,the calculated internal reorganization energies λ1CT–3LE and λ3LE–1CT are 179.6 meV and 208.8, respectively, which aremore than 350 times higher than the internal λ1CT–3CT value of 0.5 meV. The analysis ofsolvatochromic effect on the photophysical parameters revealed thatthe 1CT ↔ 3LE transition with large reorganizationenergy do not explain the experimental growth of rISC and ISC rateswith the polarity of medium. In various media from hexane to acetonesolutions, the ΔE1CT–3LE values estimated experimentally changed in the largerange from +94 to −460 meV, whereas the ΔE1CT–3CT values changed from24 to 4.2 meV. The corresponding TD-DFT calculated values were 559and 6.6 meV, respectively. In contrast to the 1CT ↔ 3LE transitions, the 1CT ↔ 3CTones perfectly explained the experimental dependencies due to muchsmaller reorganization energy and energy gap as well as vibrationallyenhanced SOC.In DMAC-TRZ, substantial differencesin geometries and dipole momentsof 3LE state in spin-flipprocesses should thus be carefully analyzed using both ΔE1CT–3LE and λ1CT–3LE values, namely, how thechange of latter values correlate with the experimental rate constants.In our opinion, the evidence of fast ISC and rISC between the 1CT and 3CT states presented here significantlychange the current understanding of TADF photophysics. Previous interpretationof the photophysical properties of TADF emitters based on the ΔE1CT–3LE value asthe key parameter and negligible rate of the 3CT ↔ 1CT transitions should be reconsidered.In D–A emitters, the role of E1CT–3CT.The main conclusion of this research is that correct descriptionof the basic photophysical parameters of TADF emitters such as ratesof ISC, rISC, and radiative deactivation is not possible without takinginto account molecular vibrations and deviation from the orthogonalityof D–A planes. A relatively simple and illustrative model presentedhere brings further important consequences. The evidence of efficientinteraction of the same CT states with different multiplicities changessignificantly the current understanding of TADF. Photophysics of someemitters as well as molecular design principles should be revisedwith an accent on Δ1CT ↔ 3CT interaction, which is forbidden in “frozen” optimalexcited-state geometry but is effectively activated by molecular vibrationsand accelerated in polar media due to the reduction of the 1CT–3CT energy gap.Experimental investigations of DMAC-TRZ insolvents of large rangeof polarity provided evidence of the increase of rates of forwardand rISC under the stabilization of CT states. Supported by the TDDFTcalculations, the polarity dependence of ISC and rISC was explainedby the growing efficiency of the 1CT and 3CT states of thesame nature. According to the theoretical and experimental results,ISC and rISC occur through the 3CT ↔ 1CT channel exclusively. The exception is the medium of lowest polarity,hexane, where the ΔE1CT–3CT value is the highest one and the ISC rate is abnormallyhigh, indicating some influence of the 3LE state.To describe correctlythe experimental data, a theoretical modelwas developed, which treats photophysical parameters as statisticalsums of respective values for selected rotational isomers existingat room temperature. Molecular vibrations changing the dihedral anglebetween donor and acceptor fragments were found to increase the SOCsharply between the 1CT ↔ 3CT transitions are the vibrationally enhanced SOC, very smallenergy gap, and comparably small reorganization energy due to similargeometries in the 1CT and 3CT states. In contrast,much larger reorganization energy λ1CT–3LE, growing |ΔE1CT–3LE| value, and decreasing populationof the 3LE state result in negligible contribution of 3LE ↔ 1CT transition rate in media more polarthan hexane.The main factors enabling effective Further development of high-precision theoreticalTADF model ata given temperature should take into account numerous molecular vibrationsand their impact on the electronic properties of lowest excited states.Moreover, special attention should be given to the effects of viscosityof medium on molecular vibrations, especially in the case of amorphousemitting layers in optoelectronic devices.


Staphylococcus aureus (SA) bacteremia (SAB), including right-sided infective endocarditis (IE), used in addition to standard-of-care antibiotics. CF-301 in vitro activity was determined against SA isolates reflecting the Phase 3 target patient SAB population, including IE.Exebacase (CF-301) is a lysin (peptidoglycan hydrolase enzyme) with anti-staphylococcal activity. CF-301 is in Phase 3 of clinical development for the treatment of 666 SA recovered from blood (3% from known IE) of patients hospitalized in 29 centers located in 9 Census regions were included as part of the SENTRY Antimicrobial Surveillance Program for 2020. Identification was confirmed by MALDI-TOF. Susceptibility to 12 comparators used reference broth microdilution (BMD), whereas CF-301 used a modified BMD method with cation-adjusted Mueller-Hinton broth (CAMHB) supplemented with 25% horse serum and 0.5 mM DL-dithiothreitol according to CLSI. MIC interpretation for comparator agents used CLSI criteria, including determination of multidrug-resistant (MDR) phenotype (non-susceptible to ≥3 classes of antibiotics).50, MIC90 and modal MIC values of 0.5 mg/L. CF-301 MIC results  against methicillin-susceptible (MSSA) and -resistant  SA were identical. Many comparators had activity against MSSA; among drugs indicated for treating SAB caused by MRSA, daptomycin and vancomycin were active (100% susceptible) against all isolates. A total of 62.3% of MRSA isolates were categorized as MDR, and CF-301 showed equal MIC50 and MIC90 results against MDR  and non-MDR  populations. Daptomycin and vancomycin were active (100% susceptible) against MDR MRSA isolates.Against all SA tested CF-301 had an MIC range of 0.06-1 mg/L, with MICCF-301 was uniformly active against contemporary SA isolates responsible for bloodstream infections in the US in 2020. CF-301 activity was consistent, regardless of resistance phenotype . Surveillance data presented here further support the clinical development of CF-301 as a promising option for treatment of SAB, including those caused by MDR MRSA isolates.Rodrigo E. Mendes, PhD, AbbVie (Research Grant or Support)AbbVie (formerly Allergan) (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)ContraFect Corporation (Research Grant or Support)GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Nabriva Therapeutics (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Jill Lindley, Bravos Biosciences (Research Grant or Support)ContraFect Corporation (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Nabina Gurung, n/a, ContraFect Corporation (Research Grant or Support) Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  Involved: Self): Research Grant or Support; BioVersys Ag  Involved: Self): Research Grant or Support; Bugworks  Involved: Self): Research Grant or Support; Cidara  Involved: Self): Research Grant or Support; Cipla  Involved: Self): Research Grant or Support; Contrafect  Involved: Self): Research Grant or Support; Cormedix  Involved: Self): Research Grant or Support; Crestone, Inc.  Involved: Self): Research Grant or Support; Curza  Involved: Self): Research Grant or Support; CXC7  Involved: Self): Research Grant or Support; Entasis  Involved: Self): Research Grant or Support; Fedora Pharmaceutical  Involved: Self): Research Grant or Support; Fimbrion Therapeutics  Involved: Self): Research Grant or Support; Fox Chase  Involved: Self): Research Grant or Support; GlaxoSmithKline  Involved: Self): Research Grant or Support; Guardian Therapeutics  Involved: Self): Research Grant or Support; Hardy Diagnostics  Involved: Self): Research Grant or Support; IHMA  Involved: Self): Research Grant or Support; Janssen Research & Development  Involved: Self): Research Grant or Support; Johnson & Johnson  Involved: Self): Research Grant or Support; Kaleido Biosceinces  Involved: Self): Research Grant or Support; KBP Biosciences  Involved: Self): Research Grant or Support; Luminex  Involved: Self): Research Grant or Support; Matrivax  Involved: Self): Research Grant or Support; Mayo Clinic  Involved: Self): Research Grant or Support; Medpace  Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd.  Involved: Self): Research Grant or Support; Melinta  Involved: Self): Research Grant or Support; Menarini  Involved: Self): Research Grant or Support; Merck  Involved: Self): Research Grant or Support; Meridian Bioscience Inc.  Involved: Self): Research Grant or Support; Micromyx  Involved: Self): Research Grant or Support; MicuRx  Involved: Self): Research Grant or Support; N8 Medical  Involved: Self): Research Grant or Support; Nabriva  Involved: Self): Research Grant or Support; National Institutes of Health  Involved: Self): Research Grant or Support; National University of Singapore  Involved: Self): Research Grant or Support; North Bristol NHS Trust  Involved: Self): Research Grant or Support; Novome Biotechnologies  Involved: Self): Research Grant or Support; Paratek  Involved: Self): Research Grant or Support; Pfizer  Involved: Self): Research Grant or Support; Prokaryotics Inc.  Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support Ray Schuch, PhD, ContraFect Corporation (Employee) Jane E. Ambler, PhD, ContraFect Corporation (Employee)


Localized malignant mesothelioma is rare. It has a histological pattern identical to diffuse malignant mesothelioma but without diffuse serosal spread. Localized malignant mesothelioma typically originates from the pleura, peritoneum or pericardium, but can occasionally develop from organs. Our cases represent what might be the largest mediastinal localized malignant mesothelioma described and the first presentation of the epithelioid type in the stomach of an adult. Localized malignant mesothelioma (LMM) is a rare neoplasm [1]. Localized malignant mesothelioma (LMM) is a rare neoplasm . The hisA 66-year-old male, previously asbestos-exposed, non-smoker, presented with 3 months of intermittent fever, fatigue, mild cough, right-sided chest pain and a weight loss of 7 kg.A positron emission tomography-computed tomography (CT) showed an 8 × 6 × 9 cm tumour along the right side of the heart Fig. . A biopsThe tumour was considered a primary lung cancer, adjacent to the mediastinum and diaphragm and thought to be primary resectable. A right-sided anterior muscle-sparing thoracotomy was performed with the intention of resecting the tumour.During exploration, the tumour was found to extend more widely than expected. The thoracic part of the tumour was located on the right side of the mediastinum, measured 10 × 6 × 6 cm and continued in a large intraabdominal component, with no involvement of the right lung. The tumour was adjacent to the pericardium and right diaphragm. Re-evaluation of the preoperative CT and positron emission tomography-CT by liver surgeon and radiologist also suggested invasion to the left liver lobe, resembling an upper gastrointestinal tumour, which perioperative biopsy could not rule out. A perioperative gastroscopy showed no involvement of the oesophagus.The procedure was terminated in order to re-evaluate and plan. The scans were consulted with a liver surgeon, and the surgical access was planned differently.Three weeks later, definitive surgery was performed: In toto resection of the tumour through a laparoscopic and midline trans-sternal approach. By laparoscopy, the tumour was dissected from the liver. There were no signs of invasion into the liver nor diaphragm from the abdomen. From the sternotomy, the mediastinal tumour including the affected pericardium and diaphragm was resected. For reconstruction of pericardium and diaphragm, 2 Goretex patches were used.Histological analysis revealed malignant epithelioid mesothelioma assumed to derive from the mediastinum.The diaphragmatic resection margins contained tumour cells and the patient underwent adjuvant chemotherapy. A regime of 6 cycles of Cisplatin/Pemetrexed was planned, but only 3 cycles were given due to neutropenia.A CT scan 6 months after surgery detected recurrent disease with an 8-mm pleuritic thickening resulting in additional chemotherapy and subsequent palliative radiotherapy due to tumour progression and symptoms of dyspnoea and thoracic pain. After the conclusion of palliative radiotherapy, 22 months after surgery, the symptoms improved. The patient has resumed his usual daily activities, e.g. able to take a bicycle ride of 30 km. CT scans show stable cancer disease over a month. The patient is now part of a project of experimental oncological treatment.A 74-year-old female, former smoker, presented with long-lasting abdominal pain, and a weight loss of 5–7 kg. CT scan Fig.  and posiGastroscopy and endoscopic ultrasound revealed an indentation in the gastric wall of the fundic area and a tumour with a diameter of 5 cm. Endoscopic ultrasound biopsy showed malignant mesothelioma, epithelioid type.Systemic neoadjuvant chemotherapy was initiated. The patient received 6 cycles of Carboplatin/Pemetrexed resulting in a 1-cm tumour regression. Adjuvant radiotherapy was not included in the treatment strategy, partially because of problematic field delimitation.A tumour adherent to the greater curvature of the stomach, diaphragm, aorta, spleen and pancreas was removed.A laparoscopic approach was used. The tumour was dissected and freed from the close adherence to the aorta and the spleen, the relation to the pancreas warranted a tangential superficial wedge resection of the body and tail of the pancreas and then closure of the pancreatic defect with absorbable self-locking thread. In order to free the tumour, a wedge resection of the involved part of the diaphragm was performed. Finally, a proximal gastrectomy dividing the oesophagus just above the cardia and the stomach on the proximal part of the corpus was performed. Reconstructing continuity of the gastrointestinal tract was done with an oesophagogastric anastomosis using a circular OrVil 25-mm stapler device. The diaphragm was closed with several single stitches and covered with a BioMesh.Postoperative histological results confirmed malignant mesothelioma of epithelioid type with clear resection margins.The patient remains recurrence-free 22 months after surgery.LMM is rare with merely 101 cases described [LMM is a serosal or subserosal, non-organ-specific tumour with a histological and immunohistochemically pattern identical to DMM, but without diffuse serosal spread , 3. ApprWhen occurring in mediastinum, the mesothelial lining cells of the pericardium are considered as the most probable cells of origin .LMM is often unexpected due to its rarity and clinical presentation, but it can occur in all ages, with males representing 70–75% of cases . AsbestoDistinguishing mesothelioma from lung adenocarcinoma is histologically challenging when using only limited tissue from core biopsies . The bioEarly and correct diagnosis between LMM and DMM is important , 4, sincWe have presented 2 unique cases: what might be the largest LMM tumour of mediastinal origin, with a rare tumour extension and disease recurrence, and the first epithelioid LMM with origin in the stomach in an adult.These cases highlight the importance of considering mesothelioma in the differential diagnosis of a solid tumour, regardless of gender, asbestos exposure and especially organ of origin.Conflict of interest: Thomas Decker Christensen has been on the speaker bureaus for AstraZeneca, Boehringer-Ingelheim, Pfizer, Roche Diagnostics, Takeda, Merck Sharp & Dohme (MSD) and Bristol-Myers Squibb and has been in an Advisory Board for Bayer and MSD. O ther authors declared no conflict of interest.