Cervical lymph node metastasis (CLNM) is common in patients with differentiated thyroid carcinoma (DTC); however, the efficiency to distinguish CLNM before surgery is limited. T cell exhaustion, characterized by the overexpression of immune checkpoints, plays a critical role in the immune evasion of tumors. The aim of this study is to analyze the association between serum levels of soluble immune checkpoints (sICs) and CLNM in DTC patients.Levels of sICs in serum of 71 DTC patients and 56 healthy volunteers were analyzed by ELISA. Peripheral blood mononuclear cells and cervical lymph nodes of DTC patients were isolated and their expression of sICs were analyzed. Lymphocytes in cervical lymph nodes were analyzed for immune checkpoints expression and transcription of exhaustion‐associated factors. 30 out of 71 DTC patients were followed up from 3 to 9 months after the operation, and postoperative sTIM‐3 were analyzed.Four sICs, including LAG‐3, PD‐1, PD‐L1, and TIM‐3, were increased in DTC patients. All four sICs exhibited higher sensitivity at discriminating CLNM than cervical ultrasound. In the patient‐matched comparison, higher sTIM‐3 levels were observed in tumor‐involved lymph nodes (TILNs) than in normal lymph nodes (nLNs). T lymphocytes in TILNs had higher TIM‐3 surface expression and increased secretion of sTIM‐3 than those in patient‐matched nLNs. Finally, postoperative serum sTIM‐3 levels were decreased in DTC patients with CLNM compared to their preoperative levels.Serum levels of sICs, especially sTIM‐3, could help to predict CLNM and provide evidence for surgical decision‐making in DTC. In this study, we analyzed serum levels of soluble immune checkpoints (sICs) and their association with CLNM in patients with DTC. We found that serum sICs analyzed in our study showed higher sensitivity and specificity in CLNM prediction than cervical ultrasound, and high sTIM‐3 levels was an independent risk factor for predicting CLNM. The results implied that serum levels of sICs, especially sTIM‐3, could help to predict CLNM and provide evidence for surgical decision‐making in DTC. As a result, tumor cells can coexist with antitumor immunity for a long time without being eliminated by immune cells, suggesting that the antitumor immune response may be compromised. T‐cell exhaustion is a special dysfunctional state due to long‐term antigen stimulation, characterized by the sustained upregulation of immune checkpoint receptor expression, gradual loss of cytotoxicity, and ultimately clonal deletion.Immune checkpoints, such as PD‐1, TIM‐3, cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4), and lymphocyte activation gene‐3 (LAG‐3), are upregulated on the surface of exhausted T cells, inhibiting the effect of T cells and driving their apoptosis. Soluble immune checkpoints (sICs) can be released via shedding of the cell membrane or alternative cleavage. The upregulation of sIC is observed in various types of tumors.In the present study, we analyzed the serum levels of sIC in DTC patients. The overexpression of sIC before treatment, especially sTIM‐3, was found to indicate a higher probability of CLNM. Further, DTC patients with CLNM had a higher proportion of TIM‐3‐positive T lymphocytes in their tumor‐involved LNs, but not in peripheral blood, and T lymphocytes in DTC‐involved LNs secreted higher levels of sTIM‐3. Finally, serum sTIM‐3 levels were found to decrease in DTC patients with CLNM after the operation. Altogether, serum sIC levels could help to predict CLNM and provide reference for surgical decision‐making in DTC.22.1This study was conducted in accordance with the Declaration of Helsinki and approved by the Human Research Ethics Committee of Qilu Hospital of Shandong University (China). All patients gave informed consent for sample collection and the subsequent analyses. Patients who were recruited had thyroid nodules detected during neck ultrasound examination and confirmed as malignant through fine‐needle aspiration (FNA) biopsy. These patients underwent thyroidectomy at Qilu Hospital of Shandong University in 2022. The choice of surgical approach follows guidelines of Chinese Society of Endocrinology and China Anti‐Cancer Association.2.2+ T lymphocytes were isolated from the single‐cell suspension of LNs using anti‐CD3‐conjugated magnetic microbeads (Miltenyi Biotech) according to the manufacturer's instructions.Preoperative serum samples were collected following laboratory testing. LNs were collected and processed according to a previously published protocol, with some modifications.The LN extract was generated for ELISA according to the protocol recommended by Abcam. Briefly, LN tissues with similar weights (approximately 5 mg) were homogenized using an electric homogenizer (Scientz‐48 L) in 800 μL complete extraction buffer . Thereafter, the homogenized tissues were centrifuged at 13,000 rpm for 20 min at 4°C. The supernatant was collected and stored at −80°C for further analysis.Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of DTC patients using gradient media (TBDsciences). The isolated PBMCs were cryopreserved as described above.2.3+ T lymphocytes from LNs were resuspended in complete RPMI‐1640 with 1:100 penicillin/streptomycin and 200 U/mL recombinant human IL‐2  and seeded in a 24‐well plate at a density of 1 × 106/mL. After 7 days, the supernatant was collected for further analysis.PBMCs or CD32.4The levels of human CTLA‐4 , LAG‐3 , PD‐1 , PD‐L1 , and TIM‐3  in serum or cell culture supernatants were quantified using commercial ELISA kits according to the manufacturer's instructions.2.5Cells isolated from LNs were stained with anti‐CD3‐PE (Elabscience), anti‐CD4‐PerCP (BD Biosciences), anti‐CD3‐PerCP (Elabscience), anti‐CD8‐PE (BD Biosciences), anti‐PD‐1‐FITC (Biolegend), or anti‐TIM‐3‐FITC (Biolegend) antibodies. The isotype controls were analyzed in parallel. The samples were processed on a FACSCalibur flow cytometer (BD Biosciences) and analyzed using the FlowJo software.2.6RNA from LNs was extracted using TRIzol Reagent (Invitrogen). cDNA was synthesized via reverse transcription. Quantitative RT‐PCR (RT‐qPCR) was performed using a QuantStudio 5 instrument (Applied Biosystems). GAPDH was used as an internal control. The primer sequences were as follows: Tbx21, forward: 5′‐CGA GAT TAC TCA GCT GAA AAT TGA T‐3′, reverse: 5′‐TGT CAA CAG ATG TGT ACA TGG ACT‐3′; Tcf7, forward: 5′‐CTG CCA TCA ACC AGA TCC T‐3′, reverse: 5′‐GCT CAT AGT ACT TGG CCT GCT‐3′; Tox, forward: 5′‐TTC TCT GTG TCA CCC CAT GA‐3′, reverse: 5′‐TCT GGC ATC ACA GAA ATG GA‐3′; and GAPDH, forward: 5′‐TCG GAG TCA ACG GAT TTG GTC GTA‐3′, reverse: 5′‐CTT CCT GAG TAC TGG TGT CAG GTA‐3′.2.7p < 0.05. Student's t‐test or Mann–Whitney nonparametric t‐test was used for statistical comparisons, depending on whether the data passed the normality test. The correlation between the two variables was determined using Pearson's correlation.All statistical analyses were conducted using SPSS software version 13.0. Statistical significance was set at 33.1The levels of sICs, including sCTLA‐4, sLAG‐3, sPD‐1, sPD‐L1, and sTIM‐3, in the preoperative serum of patients with PTC were determined by ELISA. Among the five immune checkpoints, four, except for sCTLA‐4, were detected in the serum Figure . The preV600E mutation status.We noticed an overexpression of sIC in DTC patients with CLNM Figure ; therefo3.2As the above analysis revealed that PTC patients with CLNM have higher sIC levels, we further explored whether sIC could be used as a preoperative predictor of CLNM in PTC patients. First, the ROC curve was analyzed to determine the best cutoff value for each sIC. As shown in Table Univariate analysis was then performed with the demographic characteristics and laboratory examination results of patients. As shown in Table A multivariate analysis was performed with all significant variants in the univariate analysis. Based on the results, only age and sTIM‐3 was an independent factor at predicting CLNM, with an HR of 0.08 and 48.35, respectively Table .3.3To identify the sources of sIC, we first isolated PBMCs from the peripheral blood of PTC patients with or without CLNM and cultured them in vitro. As shown in Figure 3.4+CD4+ and CD3+CD8+ T‐cell subsets of TILNs compared with patient‐matched nLNs .s Figure . In conts Figure . Consequs Figure .We proceeded to examine the expression of exhaustion‐associated transcription factors. First, we analyzed transcriptomes from the open‐access dataset, GSE60542, which contained four nLNs and 23 TILNs. Based on the results, Tbx21, Tcf7, and Tox were downregulated in TILNs Figure . However+ T cells were isolated from the TILNs and nLNs and cultured in vitro. After 7 days, sTIM‐3 was found to be significantly overexpressed in the supernatant of CD3+ T cells from TILNs compared to those from patient‐matched nLNs  than in normal LNs. Based on the expression of transcription factors, T lymphocytes in TILNs exhibited a switched exhaustion phase from the progenitor or intermediate to terminal phase, and secreted higher levels of sTIM‐3. Finally, serum sTIM‐3 levels were decreased in DTC patients with CLNM at 3–9 months after the operation.Lymph node metastasis is common in DTC, and its preoperative diagnosis is not satisfactory. T‐cell exhaustion is an important immune evasion mechanism in cancer. Previous studies revealed increased exhaustion of T cells in the peripheral blood and cervical lymph nodes of patients with DTC. In the present study, we examined the levels of sIC, whose overexpression is a characteristic of T‐cell exhaustion, in the serum of DTC patients and evaluated their association with the clinicopathological parameters of DTC. Serum sIC levels were found to be elevated in DTC patients, and their overexpression indicated a higher risk of CLNM. Among these, serum sTIM‐3 levels independently predicted CLNM. We also observed overexpression of sTIM‐3 and a higher proportion of TIM‐3The upregulation of sIC has been found in various types of tumors, including renal cell carcinoma, hepatocellular carcinoma, and non‐small‐cell lung cancer.+ and CD8+ subsets had significantly higher levels of TIM‐3 on the surface than those of nLNs of the same patient. Increased TIM‐3 expression usually reflects the later exhaustion stage of T cells.+T lymphocytes in the TILNs of DTC patients is related to their exhaustion status; however, functional experiments are needed. Importantly, the sTIM‐3 secretion levels of T lymphocytes isolated from TILNs were found to increase. Unlike other sICs, such as sGalectin‐9+ T lymphocytes in TILNs are an important source of sTIM‐3.During the exploration of sIC resources, sIC levels in LNs were found to be significantly higher than those in PBMCs, and the level of sTIM‐3 in TILNs from the same patient was higher than that in normal LNs. To our knowledge, this is the first study to report the detection of sIC levels in locoregional lymph nodes of DTC. Moreover, in TILNs with high levels of sTIM‐3, CD4Notably, a significant decrease in sTIM‐3 levels was found in the serum of DTC patients after surgical treatment with prophylactic central CLN dissection; this decrease mainly occurred in patients with CLNM. Although previous studies have shown that sICs, such as sPD‐1, remain stable or increase after certain treatments, such as radiotherapy, anti‐EGFR therapy, or immunotherapy, and is associated with better prognosis in tumor patients,In conclusion, our results suggest that sICs, especially sTIM‐3, are effective preoperative predictors of CLNM in DTC patients. The increased levels of sTIM‐3 in the serum of DTC patients with CLNM may be related to TIM‐3 overexpression on the surface of T cells in TILNs. Our study had several limitations. First, due to inaccessibility to tumor samples, we failed to isolate enough lymphocytes from DTC tissue. As a result, we could not analyze the relationship between sIC and tumor‐specific T lymphocytes in the DTC tumor microenvironment. Second, the function of sTIM‐3 is still in the preliminary exploration stage and is controversial.Yi Shao: Conceptualization (lead); investigation ; methodology ; writing – original draft . Xinru Gui: Formal analysis ; investigation (lead); methodology (lead). Yuxin Wang: Formal analysis ; investigation ; methodology . Lei Sheng: Investigation . Dong Sun: Funding acquisition ; validation (lead). Qingdong Zeng: Investigation . Huayang Wang: Conceptualization (lead); funding acquisition ; methodology ; writing – review and editing (lead).This work was supported by grants from Natural Science Foundation of Shandong Province (grant no. ZR2020LZL015 and ZR2022LZL002).The authors declare no conflict of interest.Experiments containing human materials were approved by the Human Research Ethics Committee of the Qilu Hospital of Shandong University (China). All patients have provided consent for sample collection and subsequent analyses.Data S1.Click here for additional data file.


A commonplace sight is seeing other people walk. Our visual system specializes in processing such actions. Notably, we are not only quick to recognize actions, but also quick to judge how elegantly (or not) people walk. What movements appear appealing, and why do we have such aesthetic experiences? Do aesthetic preferences for body movements arise simply from perceiving others’ positive emotions? To answer these questions, we showed observers different point-light walkers who expressed neutral, happy, angry, or sad emotions through their movements and measured the observers’ impressions of aesthetic appeal, emotion positivity, and naturalness of these movements. Three experiments were conducted. People showed consensus in aesthetic impressions even after controlling for emotion positivity, finding prototypical walks more aesthetically pleasing than atypical walks. This aesthetic prototype effect could be accounted for by a computational model in which walking actions are treated as a single category (as opposed to multiple emotion categories). The aesthetic impressions were affected both directly by the objective prototypicality of the movements, and indirectly through the mediation of perceived naturalness. These findings extend the boundary of category learning, and hint at possible functions for action aesthetics.The online version contains supplementary material available at 10.1186/s41235-023-00510-0. Living in a world full of objects and events, there is one kind of stimuli that captivates most of us: other people. Our visual systems specialize in processing sights related to other people, including their eyes Emery, , faces , bodies How do these aesthetic impressions of other people arise? Extensive research has uncovered various perceptual factors that determine facial and body attractiveness, including shape averageness, symmetry, and sexual dysmorphism  impact of appearances on our personal and professional lives, it is critical to first understand what kind of aesthetic experiences can arise upon seeing a person. This study aimed to fill the gap between the vast scientific explorations on static stimuli and dynamic real-world experiences. Second, knowledge of action aesthetics will aid the design and development of the virtual world, such as those in animations, avatars in virtual reality, and robotics. Third, being able to evaluate human movements plays critical roles in medical diagnosis and rehabilitation  from the University of California, Los Angeles (UCLA) community completed an individual 30-min experimental session online in exchange for course credit. A total of 40 additional observers participated  but were removed based on predetermined criteria (see details in the Observer exclusions section below). The sample size was predetermined arbitrarily, preregistered, and fixed throughout all experiments. The study was approved by the UCLA Institutional Review Board.Because the stimuli were rendered on observers’ own web browsers, viewing distance, screen size, and display resolutions could vary depending on computer monitors used by observers; hence, we report visual stimulus dimensions using pixel (px) values.From the motion capture dataset , they were asked to maximize their window size, informed about their task, and quizzed about their understanding of the instructions, and then, they provided their consent. During the instructions, they were shown 4 videos from different walkers and different emotional expressions, to help them gauge the range of experiences they would have. They then performed one practice trial to get familiar with the rating scale.Observers were directed to a website where stimulus presentation and data collection were controlled via custom software written in HTML, CSS, JavaScript, JQuery, and PHP. Observers were not allowed to participate using phones or tablets. After completing a CAPTCHA task . For Experiment 3, instead of aesthetic ratings, the observers provided ratings to indicate their subjective impression of each walking style’s prototypicality. To avoid jargon, we did not use the word prototypicality directly in the instructions, but asked the observers to rate the walking styles’ naturalness . Again, they used a 6-point scale with naturalness labels .In the second part of all three Experiments, the observers rated the emotion positivity of the walkers (Experiment 1 and 3) or the creatures (Experiment 2) in the videos , using a 6-point scale with emotion positivity labels .The videos displayed intact walkers in Experiment 1 and 3, and spatially scrambled walkers in Experiment 2. Each of the 80 videos was displayed once in each block in different random orders. Whether the original or the mirrored version were shown was randomly decided for each video and each observer but kept the same across the two rating tasks.At the end of the experiment, observers answered a series of debriefing questions to ensure they had completed the experiment without any issues.In addition to the 150 observers whose data were analyzed, 40 observers  participated and were excluded based on criteria decided before data collection began, with some observers triggering more than one criterion. For Experiment 1, five observers reported that they did not follow the instructions or did not take the experiment seriously; one observer failed the instruction quiz more than once; one observer spent less than 0.5 s on at least one page of the instructions; one observer had a browser viewport smaller than 800px × 600px; one observer had at least one trial with the video not fully in view during the rating task; one observer gave the same rating to more than 15 consecutive trials; six observers hid the experiment browser tab more than three times during the trials; and four observers took too long to complete the experiment .For Experiment 2, seven observers reported that they did not follow the instructions or did not take the experiment seriously; one observer spent less than 0.5 s on at least one page of the instructions; three observers had a browser viewport smaller than 800px × 600px; one observer had more than four trials with response times longer than two minutes in at least one block; two observers hid the experiment browser tab more than three times during the trials; one observer provided a non-sensical answer to one of the debriefing questions; and three observers took too long to complete the experiment.For Experiment 3, one observer reported technical issues, three observers reported that they did not understand the instructions or did not take the experiment seriously; one observer spent less than 0.5 s on at least one page of the instructions; one observer had at least one trial with the video not fully in view during the rating task; three observers hid the experiment browser tab more than three times during the trials; and two observers took too long to complete the experiment.Each video was preprocessed separately following these steps. (a) We first subtracted the head’s x position in each frame from all joints’ x positions to remove the global horizontal movement. (b) The mean x and y positions for each joint across all the frames were calculated and subtracted from the joint’s x and y positions in each frame. This way, all joints’ trajectories ended up centering at coordinate . (c) The video was segmented manually into multiple action clips based on the type of movements the walker was performing—walking from left to right, turning around on the right, walking from right to left, or turning around on the left. The number of action clips differed between videos and ranged from 1 to 5 clips.Since the same type of movements in different action clips was performed in each walker’s own walking speed and rhythm. Comparing different clips required a method to map the corresponding frames in the gait cycle across walkers. For example, the frame where Walker A raised their right foot in Clip A should be compared with the frame where Walker B raised their right foot at a different time point in Clip B. We used dynamic time warping algorithms (DTW) to measure dissimilarity between walking sequences. DTW was performed by “warping” the temporal sequences of coordinates nonlinearly in time to find the optimal  correspondence between two sequences. We use a simple example to illustrate this algorithm: Considering computing dissimilarity between two sequences of 2D coordinates—A sequence:  and B sequence: , the algorithm would correspond A1  and A2 (the second frame of A sequence) both to B1 (the first frame of B sequence), and A3 to B2, and finally A4 to B3, B4, and B5. With this correspondence mapping over time, the algorithm can minimize the total dissimilarity, calculated by the distance between matched coordinates (0 + 0 + 1 + 0 + 0 + 1 = 2). This procedure allowed us to map corresponding frames between two clips in an automatic and data-driven manner.To model prototypicality, we performed pairwise comparison between all 80 videos. For each action clip in each video, we applied DTW algorithm multiple times to find the best matching sequence in all other 79 videos following these steps: (a) We measured the clip length in frame number and dropped any action clip that had less than six frames (200ms) as they contained too little information for meaningful mapping. (b) For each of the rest of the action clips, we found the best matching sequences within the full length of each of the other 79 videos. We did so by defining the max and min clip length for the possible matching sequence as ± 12 frames from its own clip length, with the constraint that the length should be no longer than 150 frames (the full video) and no shorter than seven frames. (c) We looped through all allowed clip lengths for the possible matching sequences. For each clip length, there will be multiple possible matching sequences in each of the other 79 videos. For example, a clip length of 5 frames includes sequences like Frame 1–5, Frame 2–6, Frame 3–7, and so on, from another video. We performed DTW between the action clip and each of these possible matching sequences to find the one that had the smallest dissimilarity across clip length. Thus, we found the best corresponding sequence and its dissimilarity to the action clip for each of the other 79 videos. Note that the DTW was performed considering all 15 joints’ coordinates at the same time.The total dissimilarity between two videos was then calculated by summing over the dissimilarity of each action clip in one video and their best corresponding sequences in the other video. This dissimilarity score was then normalized to the standard video length of 150 frames (since a few action clips might be dropped from finding a corresponding sequence because of its short clip length). Note that this process yielded asymmetrical dissimilarity scores when we find the matching sequences from Video A to Video B, and Video B to Video A. We simply took the smaller value to represent two videos’ dissimilarity. A video’s prototypicality score was calculated by one divided by its average dissimilarity score with all other videos in a category . Hence, higher prototypicality score of a video indicates that the video is more representative of a category as it shows higher average similarity to all exemplars in this category.z-scores of the ratings and the residuals after regressing out emotion positivity ratings, we followed these steps: (a) We first calculated the predicted aesthetic rating for each video according to a linear regression model including both modeled prototypicality and naturalness rating as predictors. (b) The aesthetic ratings’ residuals from these predicted values were calculated. (c) We calculated the predicted naturalness rating for each video according to a linear regression model including only modeled prototypicality as the predictor. (d) The naturalness ratings’ residuals from these predicted values were calculated. (e) We iterated the following steps (f-j) for 10,000 times. (f) The residuals calculated from step b were permutated and led to a permutated set of aesthetic ratings. (g) The same permutation was applied to naturalness residuals and led to a permutated set of naturalness ratings. (h) Using a linear regression model with model prototypicality as the predictor to predict permutated naturalness ratings, we found the permutated slope for prototypicality (denoted by a*). (i) Using a linear regression model with both modeled prototypicality and the original naturalness rating as predictors to predict permutated aesthetic ratings, we found the permutated slope for naturalness (denoted by b*). (j) We then multiplied a* and b* to get the permutated indirect effect. (k) After 10,000 iterations, we got a distribution of a* x b* values and found the confidence interval based on the 250th and the 9751st values after sorting.To understand the causal path between objective prototypicality and aesthetic impression, we conducted a mediation analysis to separate direct effect and indirect effect through subjective prototypicality. Beside the standard Sobel test, we performed the “permutation confidence interval for ab” method . This pattern indicates substantial consensus across observers and systematic variations in aesthetic impressions across different walks.To gauge the consensus on aesthetic impressions, we used data from Experiment 1 and calculated each observer’s “aesthetic taste typicality”  = 0.662, p < 0.001; Scrambled creatures: r(78) = 0.627, p < 0.001; all tests reported were two-tailed tests), and further supported by a one-sample t-test of comparing correlations between the two ratings at the individual level to zero , t(49) = 12.18, p < 0.001, Cohen’s d = 1.72; with 48/50 observers showing positive correlations, p < 0.001; Scrambled creatures: M = 0.186, SD = 0.174, Range = ), t(49) = 7.59, p < 0.001, Cohen’s d = 1.07; with 42/50 observers showing positive correlations, p < 0.001).For both Experiment 1  and Experiment 2 (scrambled creatures), we depict each video’s mean aesthetic and emotion positivity ratings in Fig. Mhappy = 3.5 (0.6), Mneutral = 3.9 (0.5), Mangry = 2.9 (0.6), Msad = 3.1 (0.6); and showed a significant main effect of emotion category on aesthetic ratings  = 62.0, p < 0.001, η2p = 0.559; all post hoc comparison with neutral walkers: ts > 4.4, ps < 0.001 after Bonferroni correction; see Additional file Mhappy = 4.0 (0.4), Mneutral = 3.7 (0.4), Mangry = 3.4 (0.5), Msad = 2.6 (0.4); one-way ANOVA main effect with z-scores: F = 185, p < 0.001, η2p = 0.791; all post hoc comparison with happy walkers: ts > 5.9, ps < 0.001 after Bonferroni correction). Furthermore, the effect of neutral walkers having higher aesthetic ratings than other emotion categories of walkers remained to be significant after regressing out the emotion positivity ratings from the aesthetic ratings  = 41.0, p < 0.001, η2p = 0.455; all post hoc comparison with neutral walkers: ts > 5.8, ps < 0.001 after Bonferroni correction). In Experiment 2, when body configuration was removed by spatial scrambling, this effect was not observed , Mneutral = 3.6 (0.4), Mangry = 3.8 (0.6), Msad = 3.3 (0.5); main effect with z-scores: F = 18.7, p < 0.001, η2p = 0.276).If the effect of emotion positivity was unidimensional (differing only with respect to how positive the expressions appeared), the relationship between aesthetic and emotion positivity ratings described above would predict that happy walkers/creatures would yield most positive aesthetic impressions compared to neutral, angry, and sad walkers/creatures. However, we found a different patterns of results: In Experiment 1 with intact walkers, when different emotional categories were examined Fig. b, the neWhy do we aesthetically prefer neutral walkers? If categorical representations based on prototypes exist for human walkers, it is possible that neutral walkers appeared the most prototypical, and thus, the preference is a result of an aesthetic prototype effect. To test this idea, we used a computational model to quantify objective prototypicality in walking stimuli, and in Experiment 3 we measured observers’ subjective prototypicality.z-scores average: r(78) = 0.566, p < 0.001, aesthetic residuals average: semipartial correlation r(78) = 0.546, p < 0.001). Critically, the objective prototypicality index revealed that the neutral walkers indeed were the most prototypical  = 6.91, p < 0.001, all post hoc comparison with neutral walkers: ts > 2.9, ps < 0.05 after Bonferroni correction). Thus, the computational model provides an account for the aesthetic prototype effect, supporting the hypothesis that dynamic events involve representations of a category.We first constructed a single category model: Using the dynamic time warping algorithm (DTW) to compute the similarity of joint movements from two actions  = 0.382, p < 0.001). However, this correlation from the emotion category model was weaker than that from the single category model (emotion category model: r(78) = 0.382, single category model: r(78) = 0.566; comparison: p = 0.012; all comparisons between correlations were conducted with cocor, Diedenhofen & Musch, r(78) = 0.440, p < 0.001) numerically  = 0. 546; comparison: p = 0.138). Thus, the additional emotion-based categories worsened the model predictions of aesthetic ratings for the walkers, suggesting that the aesthetic judgments are better explained by assuming that human walks form a single category.Walking stimuli in our study might be represented as exemplars of a single category , or as exemplars of multiple categories depending on the different emotions the walkers were expressing . Would a model with more refined emotional categories more accurately predict human aesthetic judgments relative to the parsimonious model with a single category of walking? To address this question, we tested the emotion category model: For each walk, instead of computing the overall average similarity to the other 79 walks, we computed average similarity only within the same expression .After observing the strong correlation between prototypicality and aesthetic impressions, we next ask: what is the causal relationship between these variables? In general, correlational data afford multiple causal interpretations. However, in this case an additional constraint is apparent: model-derived prototypicality is an objective statistical measure that solely depends on the distribution of stimulus exemplars, whereas aesthetic impressions were measured by subjective human judgments. It seems logically impossible for a subjective measure to have a causal impact on an objective statistical measure on stimuli. A remaining question, however, is whether the causal path from objective prototypicality to aesthetic impression operates by a direct causal path, indirect path via some other variables , or both.While it is often possible in general that a third factor could cause both objective prototypicality and aesthetic experiences, it is paradoxical in this specific case: the third factor would need to influence the objective prototypicality, i.e., causing the estimate to be higher or lower, yet not itself be part of the objective prototypicality, i.e., not changing the estimate. For example, perhaps actors find it easier to perform neutral walks compared to happy, angry, and sad emotional walks, which led to differences in naturalness in the movements. However, this could not be considered as a third factor that could cause both the differences in objective prototypicality and aesthetic experiences, as the differences in naturalness manifest in the movements themselves and thus, would be part of the measure of objective prototypicality, rather than acting as a third factor that could influence the objective prototypicality.r(78) = 0.556, p < 0.001; with naturalness residuals after regressing out the emotion positivity: semipartial correlation r(78) = 0.540, p < 0.001), suggesting that typical movements were indeed associated with more natural impressions. This result was further supported by the similar patterns found with naturalness ratings , Mneutral = 4.0 (0.5), Mangry = 3.1 (0.5), Msad = 3.5 (0.5); one-way ANOVA main effect with z-scores: F = 55.6, p < 0.001, η2p = 0.531; all post hoc comparison with neutral walker: ts > 6.0, ps < 0.001 after Bonferroni correction). Moreover, the effect persisted after regressing out the emotion positivity ratings from the naturalness ratings  = 58.4, p < 0.001, η2p = 0.544; all post hoc comparison with neutral walkers: ts > 6.4, ps < 0.001 after Bonferroni correction). For emotion positivity ratings, we replicated the finding in Experiment 1 that happy walks were perceived as the most positive (Mhappy = 3.9 (0.4), Mneutral = 3.5 (0.4), Mangry = 3.4 (0.5), Msad = 2.5 (0.4); one-way ANOVA main effect with z-scores: F = 227, p < 0.001, η2p = 0.823; all post hoc comparison with happy walkers: ts > 9.0, ps < 0.001 after Bonferroni correction).We first examined the relationship between objective prototypicality derived from the single category model and subjective prototypicality  provided by observers from Experiment 3. Each video’s mean naturalness ratings and model-derived prototypicality are depicted in Fig. ngs Fig. f and modt(77) = 4.84 p < 0.001; the permutation CI = , which did not include zero; residual analyses: the Sobel test, t(77) = 5.18, p < 0.001; the permutation CI = , which did not include zero). The gross direct relationship between the objective prototypicality and the aesthetic impression  was substantially weakened after removing the indirect effect of the subjective impression of prototypicality .Next, we asked if a movement’s model-derived prototypicality exerts its effect on aesthetic experience through a subjective impression of prototypicality. We conducted mediation analyses to examine both the direct effect , and the indirect effect . Because the same stimuli were used in Experiments 1 and 3, this analysis included both aesthetic judgments (from Experiment 1) and naturalness judgments (from Experiment 3). With a Sobel test and the method of permutation confidence interval  People share substantial consensus on how aesthetically pleasing a walk looks. (2) Human walks look more aesthetically pleasing when they expressed positive emotions; this preference depends on holistic processing and explicit recognition of human body configurations. (3) Aesthetic prototype effects can be observed in human actions: People find prototypical walks more aesthetically pleasing than atypical walks. (4) This effect was caused both directly by the prototypicality of the walk itself and indirectly through the mediation of the subjective impression of prototypicality.The observed prototype effects indicate that human walks expressing different emotion states form a single category within a representational space, which opens a new dimension in the exploration of categorical processing. Beyond static objects and animals, dynamic events can form categories and afford the same kind of representational structures that lead to prototype effects. These categories likely form at the basic level (Rosch et al., At the same time, the demonstration of an aesthetic prototype effect in human walks also constitutes a unique approach to understanding human action aesthetics. In contrast to the focus on domain-specific explanations for aesthetic experiences based on dance movements (Cross et al., These discoveries inform several practical fields. The discovered clear consensus of how good a walk looks suggests a new source of bias to navigate in our personal and professional lives. At the same time, the prototype effect introduces a design principle for depicting animated humans, developing virtual agents, and creating robots. The computational models used in this study can also be used for machine assistance in medical diagnosis, rehabilitations, and prosthetic limbs design Pitkin, , such asAdditional file 1. Posthoc tests for all main effects of emotion category across three experiments and the modeling results were reported here.


The PINNACLE FLX  trial evaluated the safety and efficacy of a next‐generation left atrial appendage closure device . At 1 year, the study met the primary end points of safety and anatomical efficacy/appendage closure. This final report of the PINNACLE FLX trial includes the prespecified secondary end point of ischemic stroke or systemic embolism at 2 years, also making it the first report of 2‐year outcomes with this next‐generation left atrial appendage closure device.2DS2‐VASc score ≥2 (men) or ≥3 (women), with an appropriate rationale for left atrial appendage closure, were enrolled to receive the left atrial appendage closure device at 29 US centers. Adverse events were assessed by an independent clinical events committee, and imaging was assessed by independent core laboratories. Among 395 implanted patients , the secondary efficacy end point of 2‐year ischemic stroke or systemic embolism was met, with an absolute rate of 3.4%  and an upper 1‐sided 95% confidence bound of 5.3%, which was superior to the 8.7% performance goal. Two‐year rates of adverse events were as follows: 9.3% all‐cause mortality, 5.5% cardiovascular death, 3.4% all stroke, and 10.1% major bleeding (Bleeding Academic Research Consortium 3 or 5). There were no additional systemic embolisms, device embolizations, pericardial effusions, or symptomatic device‐related thrombi after 1 year.Patients with nonvalvular atrial fibrillation with CHAThe secondary end point of 2‐year stroke or systemic embolism was met at 3.4%. In these final results of the PINNACLE FLX trial, the next‐generation WATCHMAN FLX device demonstrated favorable safety and efficacy outcomes. P<0.0001) below the prespecified performance goal of 4.21%. The primary effectiveness end point of effective LAAC at 1 year was 100% (n=342), with a lower 1‐sided 95% CI of 99.1%, which was significantly above the prespecified performance goal of 97.0% (P<0.0001). At 1 year, 90% of patients had no detectable peridevice leak and, among those with any degree of residual peridevice leak, all jet sizes were measured as ≤3 mm.Transcatheter left atrial appendage closure (LAAC) is recommended as a nonpharmacologic treatment option for the prevention of thrombotic stroke in patients with nonvalvular atrial fibrillation (AF) who have contraindications to long‐term oral anticoagulation.These final 2‐year results of the trial represent the first report of longer‐term outcomes with this next‐generation LAAC device.https://www.bostonscientific.com/en‐US/data‐sharing‐requests.html).The PINNACLE FLX  trial is a single‐arm, prospective, nonrandomized trial designed to evaluate the safety and performance of this next‐generation WATCHMAN FLX LAAC device. Materials and methods have been previously described in detail2DS2‐VASc score of ≥2 for men or ≥3 for women, were able to take the prescribed postimplant antithrombotic medication regimen, had a rationale for a nonpharmacologic approach to stroke prevention, and had no other diagnoses that would require long‐term anticoagulation. Institutional Review Board approval was obtained at all sites, and all patients gave written informed consent before enrollment.A total of 400 patients were enrolled across 29 investigational centers in the United States. Patients were eligible for enrollment if they had nonvalvular AF and a CHAAdverse events were adjudicated by an independent clinical events committee (CEC), and imaging was independently assessed by a core laboratory , CAP (Continued Access to PROTECT‐AF Registry), PREVAIL, and CAP2 (Continued Access to PREVAIL AF Registry) studies.For the secondary effectiveness end point of 2‐year CEC‐adjudicated ischemic stroke or systemic embolism, a performance goal of 8.7% was established. This value was based on an expected rate of 4.7%, as observed in PREVAIL ‐eligible subjects with a CHADS2DS2‐VASc score was 4.2±1.5, and the mean HAS‐BLED score was 2.0±1.0.Baseline demographics and procedural characteristics have been previously published,The next‐generation LAAC device was successfully implanted in 395 (98.8%) patients, defined as the successful delivery and release of the LAAC device in the correct position in the LAA. In the 5 patients who did not have successful implantation, 3 were attributable to unsuitable anatomy , and 2 were attributable to inadequate final device compression and/or LAA seal. The most common device size implanted was 27 mm (31.1%); 11.4% of patients received a 20‐mm device, and 7.8% received a 35‐mm device; note that neither of these extreme sizes (20 and 35 mm) was previously available with the first‐generation device Table .Two‐year clinical follow‐up was completed for 96.9% (373 of 385) patients. A total of 15 patients withdrew by 2 years: (1) 5 patients who were not successfully implanted were not followed up beyond 45 days per protocol, and (2) 10 additional patients withdrew consent. An additional 12 patients missed the 2‐year clinical visit with no subsequent clinical follow‐up.Medication use at 1 and 2 years is shown in the At 2 years, the CEC‐adjudicated rate of ischemic stroke or systemic embolism was 3.4% , with a 95% upper 1‐sided confidence bound of 5.3%. This was below the performance goal of 8.7%; therefore, the end point was met . The ratClinical outcomes at 1 and 2 years are shown in the The next‐generation WATCHMAN FLX was designed to address several limitations of the predicate device, including reduced pericardial effusions, improved LAA sealing, and enhanced device stability. The primary results of the PINNACLE FLX trial demonstrated excellent procedural safety and substantially lower peridevice leak compared with prior studies. Whether these early findings translate into improved longer‐term clinical outcomes has not been evaluated.These final results of the PINNACLE FLX US approval trial demonstrate the sustained safety and efficacy of the next‐generation LAAC device through 2 years. The prespecified secondary end point of long‐term ischemic stroke/systemic embolism was met, with a rate of 3.4%  and an upper 95% confidence bound of 5.3%, which was better than the performance goal of 8.7%. In longer‐term outcomes, between 1 and 2 years, 1 additional patient experienced an ischemic event, and no additional device related thrombus or pericardial effusions requiring intervention were reported. No device embolizations were reported throughout the entire study.2DS2VASc scores. Two‐year all‐cause mortality rates in other device arms of LAAC studies with baseline CHA2DS2VASc score of 4.0 to 4.5 have ranged from 7.6% to 16.4%,As published previously, similarly low ischemic stroke rates per 100 patient years were noted after LAAC with the legacy first‐generation LAAC device.Prior studies suggest that peridevice leak may be associated with ischemic stroke.The infrequent occurrence of ischemic stroke beyond 1 year, only 1 additional patient had an ischemic stroke event between year 1 and 2, may be related to the enhanced rate of anatomic closure, although this remains speculative, as the study design was observational and the sample size relatively small to robustly define the rates of ischemic stroke and systemic embolism. This remains to be conclusively proven.ClinicalTrials.gov Identifier: NCT04394546).When comparing OACs, with the exception of dabigatran, ischemic stroke rates were similar between the other direct OACs and warfarin. The safety and long‐term clinical efficacy of the WATCHMAN FLX device compared with direct OACs in a contemporary population of OAC‐tolerant patients is underway and will be established by the WATCHMAN FLX Versus NOAC for Embolic ProtectION in the Management of Patients With Non‐Valvular Atrial Fibrillation (CHAMPION‐AF) randomized trial  trial was sponsored and funded by Boston Scientific, Inc .Shephal K. Doshi is a consultant to Boston Scientific, Biosense Webster, Abbott Vascular, and Conformal Medical; and is coprincipal investigator of the PINNACLE FLX, CHAMPION AF, and Conform IDE trials. Saibal Kar is a consultant to Boston Scientific, Abbott Vascular, Medtronic, and Laminar; and is coprincipal investigator of the PINNACLE FLX and CHAMPION AF trials. Ashish Sadhu reports consulting honoraria from Boston Scientific. Rodney Horton reports advisory board participation with Boston Scientific, Biosense Webster, and Abbott; and consulting honoraria from Boston Scientific. Jose Osorio reports Boston Scientific advisory board participation; consulting honoraria from Boston Scientific, Biosense‐Webster, Medtronic (institution), and Galaxy Medical; and research grants from Boston Scientific, Biosense‐Webster, and Medtronic (institution). Christopher Ellis reports research funding paid to Vanderbilt Medical Center from Medtronic, Atricure, Boehringer Ingelheim, and Boston Scientific; and consulting honoraria from Abbott Vascular, Atricure, Boston Scientific, and Medtronic. James Stone, Jr, reports reimbursement for proctoring and physician training from Boston Scientific; and funding for clinical research from Boston Scientific, Johnson & Johnson, and Abbott Vascular. Manish Shah reports consulting honoraria from Boston Scientific. Srinivas R. Dukkipati reports a research grant from Biosense Webster; and equity in Farapluse and Manual Surgical Sciences. Stuart Adler reports consulting honoraria from Medtronic. Devi G. Nair reports consulting honoraria and research grants from Boston Scientific, Abbott, Johnson & Johnson, and Medtronic. Jamie Kim reports consulting and speaker's honoraria from Boston Scientific, Conformal Medical, and Janssen. Oussama Wazni reports consulting and speaker's honoraria from Boston Scientific and Biosense Webster; and is principal investigator of the OPTION trial. Matthew J. Price reports consulting and speaker's honoraria from Astra‐Zeneca, Abbott Vascular, Boston Scientific, Chiesi USA, W.L. Gore Medical, and Medtronic; and research grants (to institution) from Daiichi Sankyo. Robert Shipley reports salary and equity (Boston Scientific). Thomas Christen reports salary and equity (Boston Scientific). Dominic J. Allocco reports salary and equity (Boston Scientific). Vivek Y. Reddy is an unpaid consultant to Boston Scientific; disclosures with medical companies include: Abbott, Axon, Biosense‐Webster, Biotronik, Boston Scientific, Cardiofocus, Cardionomic, CardioNXT/AFTx, EBR, Impulse Dynamics, Medtronic, Philips, Stimda, and Thermedical (consultant); Ablacon, Acutus Medical, Affera, Apama Medical, Aquaheart, Autonomix, Backbeat, BioSig, Circa Scientific, Corvia Medical, East End Medical, EPD, Epix Therapeutics, EpiEP, Eximo, Farapulse, Fire1, Javelin, Keystone Heart, LuxCath, Medlumics, Middlepeak, Nuvera, Valcare, and VytronUS ; and Manual Surgical Sciences, Newpace, Surecor, and Vizara (equity). David R. Holmes, Jr, has no disclosures to report.Data S1Table S1Click here for additional data file.


Chronic rhinosinusitis with nasal polyposis (CRSwNP) is a typical type‐2 inflammation involving T‐helper type‐2 cells and impairing quality of life due to nasal obstruction, discharge and reduced sense of smell. Recently, the anti‐IL4Rα antibody dupilumab was approved for CRSwNP. While dermatologic side effects in patients treated with dupilumab for atopic dermatitis are frequently observed, there is limited knowledge about these effects in patients with CRSwNP. We aimed to investigate frequency and characteristics of dermatologic side effects following initiation of dupilumab treatment in a cohort of Austrian CRSwNP patients. Therefore, CRSwNP patients presenting at the Department of Otorhinolaryngology, Head and Neck Surgery at the Vienna General Hospital were retrospectively evaluated for newly developed skin eruptions while under dupilumab treatment. Incidence was calculated and details on clinical symptoms were collected. One hundred and ninety‐two CRSwNP patients receiving dupilumab treatment were included, comprising a cumulative follow‐up of 89.65 years . We observed dermatologic side effects in four patients starting at a median time of 15.5 (range 4–23) weeks after dupilumab initiation corresponding to an incidence‐rate of 4.46  events per 100 patient‐years follow‐up. The majority  of affected patients developed psoriasis‐like dermatitis, whereas one individual experienced rosacea‐like folliculitis and alopecia areata. While dupilumab dosing was reduced in 3/4 CRSwNP patients, one patient completely stopped dupilumab therapy. Our study provides the first comprehensive evaluation of both frequency and characteristics of dermatologic side effects caused by dupilumab in CRSwNP patients. All affected patients developed Th1‐inflammatory associated skin disorders – previously observed only in individuals with prior affections of the skin (i.e. atopic dermatitis). Thus, individuals receiving dupilumab for CRSwNP may develop novel symptoms that require interdisciplinary management. Future studies on dupilumab in a real‐world setting will be required to further explore its spectrum of side effects. Onset‐date of CRSwNP, data on comorbidities as well as new onset of skin lesions were collected. When patients experienced a new onset of skin disease while under dupilumab treatment, they were referred to the department of dermatology for further assessments and diagnosis. If onset of skin eruptions occurred exclusively after dupilumab initiation, affected patients were considered to have a dermatologic side effect. Incidence rate and 95% confidence interval (95%CI) of dermatologic side effects during follow‐up was calculated.2.2This study was conducted in accordance with the 1964 Declaration of Helsinki and had been approved by the ethics committee of the Medical University of Vienna (No. 2222/2021). Due to the retrospective design, the need for an informed consent had been waived. The patients pictured in this manuscript have given written informed consent to publication of their case details.3N = 122; female, N = 77) received systemic treatment for CRSwNP of which 96% (192/199) were started on dupilumab  events per 100 patient‐years (PY). The median follow‐up was 5.5 months (IQR 5.9) and the median time from dupilumab initiation to onset of dermatologic side effects was 15.5 (range 4–23) weeks.3.1All patients showed a significant improvement of CRSwNP upon dupilumab initiation Table . Patient4This study aimed at analyzing dermatologic side effects in 192 patients receiving dupilumab for CRSwNP in a real‐world setting. Dermatologic eruptions occurred at an incidence‐rate of 4.46 per 100PY and after a median time of 15.5 weeks after dupilumab initiation. Three of four affected patients developed a psoriasiform dermatitis.Previous studies reported various adverse events related to dupilumab for CRSwNP occurring in phase‐II and phase‐III clinical trials. However, skin‐related adverse events (non‐injection site reactions) were not reported.For AD, it was previously hypothesized that blocking IL‐4Rα results in inhibition of skin‐resident Th2‐mediated inflammation thereby stimulating opposing Th1 and Th‐17 pathways. This is of particular interest, since CRSwNP like AD is a classical Th2‐mediated disease. As psoriasis is a typical Th1/Th17‐mediated skin disorder, tipping the immunological scale towards a Th1/Th17 phenotype may result in activation of a psoriasis‐specific inflammatory cytokine cascade and eventually in clinical psoriasis‐like inflammation.Strong aspects of our study comprise that it was conducted in a real‐world setting, the longitudinal follow‐up and, importantly, the detailed patient characterization. Limitations of this study include its retrospective nature, the consecutive risk to be affected by bias and the limited number of patients.In conclusion, dermatologic side effects in patients receiving dupilumab for CRSwNP were scarce and while the spectrum of immunologic modulation caused by dupilumab is still not entirely understood, further multidisciplinary surveillance is needed to elucidate its impact on type‐2 mediated‐ inflammation.DC served as a speaker and/or consultant and/or advisory board member for AbbVie, Gilead, and MSD, and received travel support from AbbVie, MSD, ViiV Healthcare and Gilead. TB has received personal fees as a consultant for Novartis. FB, AT has nothing to disclose. TQ served speaker and/or consultant for AbbVie, Novartis and Sanofi. TQ is a sub investigator for Abbvie, Elli Lilly, Galderma, Novartis. JED served as a speaker and/or consultant and/or advisory board member from Sanofi, Allergopharma, Astrazeneca, GSK and Novartis. JED is an investigator for Novartis and AstraZeneca (grants paid to her institution). SS served as a speaker and/or consultant and/or advisory board member from Sanofi, GSK and Novartis. SS is an investigator for Novartis and AstraZeneca (grants paid to his institution). CB has received personal fees as a speaker and/or consultant and/or advisory board member from AbbVie, Astra Zeneca, Bayer, Celgene, Eli Lilly, LEO Pharma, Mylan, Novartis, Pfizer, and Sanofi. CB is an investigator for Abbvie, Elli Lilly, Galderma, Novartis, Pfizer, LEO Pharma (grants paid to her institution).Appendix S1Click here for additional data file.


Patients were categorized into TMB‐high  and non‐TMB‐high  groups. Binary logistic regression analyses were performed to determine independent predictors of TMB‐high. Univariate and multivariate linear regression analyses were performed to determine independent predictors of TMB level on a log scale. Subgroup analyses for adenocarcinoma (ADC), ADC with EGFR+, ADC with EGFR−, and squamous cell carcinoma (SCC) were performed.In this retrospective study, we enrolled 129 patients with NSCLC  who underwent baseline TMB and STM tests and peak, SULmax, SULmean, MTV, and TLG) were significantly higher in the TMB‐high group than the non‐TMB‐high group; smoker , EGFR+ , KRAS+ , high CEA , and high CA125  were independent predictors of TMB‐high; and all MPs showed significant positive linear correlations with TMB on a log scale, with SULpeak as an independent predictor. However, no significant correlation was observed for SCC.For ADC, all MPs (SULMPs and STMs can predict the TMB level for patients with ADC, and may serve as potential substitutes for TMB with increased value and easy implementation in guiding immunotherapy through noninvasive methods. Unfortunately, the response rate to ICI monotherapy is only 15%–20% for unselected patients.18F‐fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) is a significant imaging modality. Previous studies have reported that baseline metabolic parameters (MPs) of NSCLC may be predictive factors for response to immunotherapy.As a functional noninvasive imaging modality based on glucose metabolism, max) and TMB in various tumor types.max and carcinoembryonic antigen (CEA) were independent predictors of TMB in NSCLC.To date, few studies have explored the correlations between MPs, STMs, and TMB,To complement the deficiency in the extant literature, we aimed to further explore the correlations of MPs and STMs with TMB. Detailed analyses of different pathological types and driver gene mutation types were performed. We expect to provide new insights for potential substitutes to TMB which has clinical utility.22.1We enrolled 2597 patients with lung cancer who underwent PET/CT examinations with data retrieved from the PET/CT Center data system between March 2018 and September 2022. The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Institutional Ethics Committee of our hospital. The inclusion criteria were as follows: (a) pathologically confirmed NSCLC; (b) baseline TMB data of the primary tumor tissue on biopsy/surgery samples obtained before treatment, excluding TMB data of metastases and lymph nodes; (c) baseline PET/CT scan performed before treatment; (d) time interval between biopsy/surgery and PET/CT scan was within 1 month to ensure an acceptable temporal correlation between imaging and genomic evaluation; and (e) measurable and describable tumor lesions on PET/CT. When a patient had multiple primary lung cancers, the lesion undergoing biopsy/surgery for TMB test was included in the analyses.Finally, 129 patients who underwent TMB tests and PET/CT scans before treatment were enrolled in the study. Clinical and demographic characteristics were retrospectively collected from medical records. We further collected baseline STM data of the enrolled patients, and the time interval between biopsy/surgery and the STM test was within 1 month. Ultimately, 98 of the 129 enrolled patients had available STM data.2.218F‐FDG at a dose of 3.70–4.44 MBq/kg. Three‐dimensional (3D) whole‐body PET/CT scans with CT were acquired approximately 50–70 min after 18F‐FDG injection. PET images were obtained in 3D mode at a rate of 2 min per frame from head to groin  and reconstructed using the VPFX‐S algorithm, with two iterations, 24 subsets, and a 4 mm Gaussian postfilter. Spiral CT scans were performed using a standardized protocol  without intravenous contrast administration. All patients underwent a breath‐hold thoracic spiral CT scan at 120 kV, 205 mA, 0.5 s per tube rotation, and a thickness of 5 mm after the PET/CT scan. Images were reconstructed from a thickness of 1.25 mm and 0.8 mm space.All patients underwent a whole‐body PET/CT scan as per the standard protocol of our hospital. Head‐to‐groin imaging was performed using an integrated PET/CT device . Patients were asked to fast for at least 6 h before the scan, and blood glucose level  was determined. Patients were intravenously injected with 2.3PET images were analyzed using PETVCAR (PET Volume Computerized Assisted Reporting), an automated segmentation software system on an Advantage Workstation . The volume of interest (VOI) of the whole tumor was autocontoured and segmented using a 3D cube. The VOI was placed by one radiologist (Q.Z.) and verified by another experienced radiologist (X.L.T.) to ensure that the 3D cube contained all FDG PET‐positive areas of the primary tumor in the axial, coronal, and sagittal planes. Disagreements were resolved by consultation. The two radiologists were blinded to the clinical data of all patients when placing the VOI.max and SUVmean within the target volume with a weighting factor of 0.5.max, SULmean, SULpeak, metabolic tumor volume (MTV), and total lesion glycolysis (TLG). SULmax was defined as the maximum SUL derived from the single voxel with the highest uptake; SULmean was calculated as the sum of SUL in each voxel divided by the number of voxels within the target volume; SULpeak was defined as the mean SUL value measured in a 1 cm3 volume spherical region of interest centered around the hottest point of the tumor; MTV represented the volume of the contoured tumor tissue with active FDG uptake; and TLG was obtained by multiplying SULmean by MTV.PETVCAR is an automated segmentation software system that uses an iterative adaptive algorithm to detect the threshold level. It separates the target volume from the background tissue by weighting SUV2.4Based on clinical applications, STMs, including CEA, carbohydrate antigen 125 (CA125), cytokeratin 19 fragment (Cyfra21‐1), and squamous cell‐associated antigen (SCC‐Ag), were tested using immunoelectrochemistry according to institutional standard laboratory procedures. Upper limits of normal were 5.0 ng/mL for CEA, 35.0 U/mL for CA125, 3.3 ng/mL for Cyfra21‐1, and 1.5 ng/mL for SCC‐Ag, respectively. Records were obtained from the database and categorized as high or normal based on the upper limits.2.5EGFR) mutation status, and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation status. Records were obtained from the official reports of the Pathology Department at our hospital. TMB was measured as mutations per megabase pair. Patients were categorized into TMB‐high (≥10 mutations/Mb) and non‐TMB‐high (<10 mutations/Mb) groups. Currently, there is no generally accepted cutoff value to define TMB‐high. In this study, TMB‐high was defined as ≥10 mutations/Mb as this category is consistent with those used in several randomized trials in lung cancerThe approach used for obtaining pathological specimens was determined according to the patient's condition: 26 patients underwent surgery, 67 underwent CT‐guided lung biopsy, and 36 underwent bronchofibroscopy biopsy. Next‐generation sequencing was used to detect the TMB level, epidermal growth factor receptor . TMB, MTV, and TLG were required to transform into the shifted log scale in the linear regression analyses because of the highly skewed distribution. Significant variables in the univariate linear regression were included in the multivariate linear regression model with backward stepwise selection.Descriptive statistics were used to analyze the differences between the TMB‐high and non‐TMB‐high groups. Categorical data were presented as numbers (percentages). The Kolmogorov–Smirnov test was used to analyze the distribution normality of continuous data. Normally distributed data were presented as mean ± standard deviation, whereas non‐normally distributed data were presented as median (range). Comparisons between the TMB‐high and non‐TMB‐high groups were evaluated using Pearson's chi‐squared test or Fisher's exact test for categorical variables and independent samples max, SULmean, and TLG) were excluded, and two variables (SULpeak and MTV) were included to construct the multivariate linear regression analyses.Notably, there was significant collinearity among PET parameters. Significant explanatory variables in multivariate linear regression analyses were identified using a backward stepwise elimination method. Variables with collinearity were not retained. The final regression model was checked to make sure each variable made a significant contribution. Multicollinearity analyses showed that the variance inflation factors for all enrolled variables were below 5.0. Finally, through model selection, three variables  were performed. Statistical analyses were conducted using SPSS . Statistical significance was set at p < 0.05.Subgroup analyses for adenocarcinoma (ADC), ADC with 33.1EGFR+ and 45.9% (45/98) were EGFR−. Patients in the TMB‐high group accounted for 20.9% (27/129) and those in the non‐TMB‐high group accounted for 79.1% (102/129), with a median TMB of 13.0 and 3.2 mutations/Mb, respectively. The values of the MPs were 8.3 ± 4.1, 11.1 ± 5.1, 5.2 ± 2.3, 15.8 (0.4–238.0), and 75.7 (0.7–2052.5) for SULpeak, SULmax, SULmean, MTV, and TLG, respectively. Ninety‐eight of the 129 enrolled patients had available STM data. The percentage of high STMs was 45.9% (45/98), 29.6% (29/98), 56.1% (55/98), and 17.3% (17/98) for CEA, CA125, Cyfra21‐1, and SCC‐Ag, respectively. Clinical information is summarized in Tables Based on the inclusion criteria, 129 patients with NSCLC, all of whom had baseline TMB and PET/CT scans before treatment, were enrolled in the present study. Among the 129 enrolled patients , 76.0% (98/129) had ADC and 24.0% (31/129) had SCC. Regarding ADC, 54.1% (53/98) were 3.2EGFR−, and SCC, respectively. Subgroup analysis of ADC with EGFR+ was not performed because only one patient had TMB‐high in this subgroup.Tables EGFR− for NSCLC, and male, smoker, EGFR−, and KRAS+ for ADC . TMB‐high was additionally associated with high CA125, Cyfra21‐1, and SCC‐Ag for NSCLC, and high CEA, CA125, and Cyfra21‐1 for ADC . All MPs  were significantly higher in the TMB‐high group than in the non‐TMB‐high group for NSCLC and ADC . In the subgroup analysis of ADC with EGFR−, TLG (p = 0.048) was significantly higher in the TMB‐high group than in the non‐TMB‐high group. However, no significant correlation was observed for SCC.TMB‐high was associated with male, SCC, smoker, and p = 0.023), EGFR+ , and high CA125  were independent predictors of TMB‐high for NSCLC, and smoker , EGFR+ , KRAS+ , high CEA , and high CA125  were independent predictors of TMB‐high for ADC.The results of the binary logistic regression analyses for NSCLC and ADC are shown in Figure 3.3EGFR+, ADC with EGFR−, and SCC, respectively. All MPs showed significant positive linear correlations with TMB (lg) for NSCLC, ADC, ADC with EGFR+, and ADC with EGFR− . However, no significant correlation was observed for SCC.Owing to the highly skewed distribution of TMB, linear regression analysis was used to identify independent predictors of TMB on a log scale. Table β = 0.212, p = 0.010), ADC , EGFR+ , and SULpeak  were independent predictors for NSCLC; age , EGFR+ , high CEA , and SULpeak  were independent predictors for ADC; SULpeak  was an independent predictor for ADC with EGFR+; and age , high CEA , and SULpeak  were independent predictors for ADC with EGFR−. SULpeak was an independent predictor of TMB (lg) for all the above categories . Figures peak and TMB (lg) for all the above categories, and no significant correlation was observed for SCC.Figure 4peak was an independent predictor of TMB (lg), including subgroups of ADC with EGFR+ and EGFR−. However, no significant correlation was observed for SCC. We identified that MPs and STMs can predict the TMB level for patients with ADC, and may serve as potential substitutes for TMB with increased value and easy implementation in guiding immunotherapy through noninvasive methods.Correlations between MPs, STMs, and TMB remain poorly understood. This study revealed different findings between ADC and SCC. For ADC, pretreated MPs and STMs were generally higher in the TMB‐high group than in the non‐TMB‐high group; all MPs showed significant positive linear correlations with TMB (lg), and SULEGFR mutation was performed for the first time, as we believed it was important that patients with EGFR− were the target candidates for immunotherapy; (b) correlations between SUL parameters and TMB were analyzed for the first time. SUL parameters eliminate the influence of adipose tissue compared to SUV parameters. PERCIST 1.0 also recommends SULpeak as the most important quantitative indicator of therapeutic response for immunotherapy; (c) CA125 and SCC‐Ag were studied for the first time, which enriched the blood‐based biomarkers; (d) TMB‐high was defined as TMB ≥10 mutations/Mb as it may be the optimal and universal cutoff to guide clinical applications; and (e) we set a 1‐month interval between PET/CT scan, STM test, and biopsy/surgery to ensure an acceptable temporal correlation. A longer time lapse may result in a poor correlation.The present study had the following improvements over previous studies: (a) subgroup analysis of the max and SULpeak were positively correlated with the degree of pathological regression to neoadjuvant immunotherapy in NSCLC , and a large majority of the patients  had SCC.max and PD‐L1 expression in SCC.This study demonstrated that TMB‐high in NSCLC was associated with male, smoker, and SCC, which was consistent with the result of previous studies.EGFR+ and TMB, which may explain why EGFR+ patients had a poor response to ICIs and even underwent hyperprogression.EGFR+ in this study, only one patient had TMB‐high , which was contradictory to the result of the negative correlation. However, the patient also had KRAS+. KRAS+ was found to be a predictor of TMB‐high and positively correlated with TMB in the univariate linear analysis. Previous studies have also reported that patients with KRAS+ had better immunotherapy efficacy.KRAS+ may be an important predictor of TMB‐high, which needs further verification.A negative correlation was observed between peak, SULmax, SULmean, MTV, and TLG) were significantly higher in the TMB‐high group than in the non‐TMB‐high group, and all MPs presented significant positive correlations with TMB. However, the exact mechanism between FDG uptake and TMB remains unclear. The mechanism of FDG uptake involves glucose metabolism, angiogenesis, hypoxia, and the mammalian target of rapamycin (mTOR) signaling pathway.We found that all MPs ; formal analysis ; methodology ; resources ; software ; visualization ; writing – original draft (lead). Xiuli Tao: Conceptualization ; data curation ; formal analysis ; funding acquisition (lead); investigation ; methodology ; project administration ; supervision ; writing – review and editing . Pei Yuan: Data curation ; formal analysis ; methodology ; resources ; software . Zewei Zhang: Data curation ; formal analysis ; methodology ; resources ; software ; visualization . Jianming Ying: Formal analysis (supporting); methodology (supporting); resources (supporting); software (supporting). Lei Guo: Formal analysis (supporting); methodology (supporting); resources (supporting); software (supporting). Ning Li: Formal analysis (supporting); methodology (supporting); resources (supporting); software (supporting). Shuhang Wang: Formal analysis (supporting); methodology (supporting); resources (supporting); software (supporting). Jing Li: Data curation (supporting); formal analysis (supporting); methodology (supporting); resources (supporting); software (supporting). Ying Liu: Data curation (supporting); formal analysis (supporting); methodology (supporting); resources (supporting); software (supporting). wei guo: Data curation (supporting); formal analysis (supporting); methodology (supporting); resources (supporting); software (supporting). Shijun Zhao: Conceptualization ; methodology ; project administration ; supervision ; writing – review and editing . Ning Wu: Conceptualization ; data curation ; funding acquisition (supporting); project administration (lead); supervision (lead); writing – review and editing (lead).The authors declare that they have no conflicts of interest.The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the Institutional Ethics Committee of the Cancer Hospital, Chinese Academy of Medical Sciences. The requirement for informed consent was waived because of the retrospective and anonymous nature of the study.


The benefit of surfactant replacement therapy for respiratory distress syndrome (RDS) has been demonstrated. However, some surfactants are expensive and usually inaccessible. Consequently, the Iranian Survanta was produced, but its effect on complications and mortality of RDS is unknown. This study aimed to compare the therapeutic outcomes of Iranian surfactant (beraksurf) and BLES (bovine lipid extract surfactant) on RDS treatment among preterm neonates.This triple blinded randomized controlled trial study was performed on 128 eligible neonates diagnosed with RDS in Afzalipour hospital in Kerman, Iran. Diagnosis of RDS, gestational age of 28–34 weeks and weight ≥ 1 kg were considered as inclusion criteria. Congenital anomalies such as congenital cyanotic heart diseases, digestive system anomalies and chromosome abnormalities were the exclusion criteria Neonates were randomly assigned into two equal groups: (1) those treated with BLES (n = 64) and (2) those treated with beraksurf (n = 64). Complications including patent ductus arteriosus (PDA), sepsis, intraventricular hemorrhage (IVH), pneumothorax, pulmonary hemorrhage, mortality, and also, the number of days required for invasive mechanical ventilation (using ventilator) and non-invasive continuous positive airway pressure (CPAP) were evaluated for all neonates. The risk ratio (RR) was calculated at 95% of confidence intervals (CI).Compared with BLES group, the RR estimate among neonates in beraksurf group was 0.89 (0.66–1.20) for PDA, 0.71 (0.23–2.13) for IVH, 0.44 (0.14–1.36) for sepsis, 0.35 (0.13–0.93) for pneumothorax, 0.33 (0.12–0.86) for pulmonary hemorrhage, and 0.55 (0.28–1.05) for mortality.Despite advances in the use of exogenous surfactants for the treatment of neonatal respiratory distress syndrome; There are still some controversial topics in this field. The results obtained in the present study showed that the two types of surfactant (BERAKSURF and BLES) have similar efficacy for the treatment and short-term outcomes in preterm infants with respiratory distress syndrome. Therefore, due to the cost-effectiveness of BRAKSURF compared to BLES, We recommend choosing BERAKSURF in terms of treatment.The online version contains supplementary material available at 10.1186/s12887-023-04406-2. Respiratory distress syndrome (RDS) is a prevalent cause of morbidity and mortality in premature neonates  usually Normally, surfactant is present in the amniotic fluid from the 28th week of gestation, and its production depends on normal pH, temperature, and perfusion . Some coSo far, there are many studies that have evaluated the effect of natural and synthetic surfactants on RDS treatment . The surA clinical trial in the United States comparing the effect of survanta surfactant and curosurf in premature babies with an average gestational age of 26–29 weeks and an average weight of 1400 g, reported a decrease in supplemental oxygen in babies receiving curosurf as well as the prevalence of patent ductus arteriosus in babies receiving curosurf Compared to surventa, it was reported less with a statistically significant difference [Another clinical trial was conducted in Zika-free NICU of Turkey in infants less than 37 weeks gestational age from July 2008 to June 2009. 126 premature babies with RDS in two groups had similar basic demographic characteristics; In this study, the group receiving survanta surfactant required two or more doses compared to treatment with Corosurf; Also, extubation in the corosurf group was more within the first three days of drug administration, and the need for Fio2 after treatment on the first, third and fifth days was significantly lower in the corosurf group. Although the rate of mortality and morbidity of the two groups were similar, the findings of the study indicated an increase in the chance of survival in the corosurf group .This clinical trial study is designed to compare the effect of the Canadian BLES with the Iranian Survanta as an efficient surfactant.This triple-blind randomized clinical trial study was performed on hospitalized neonates in neonatal intensive care unit (NICU) of Afzalipour hospital  between April 1, 2020 and February 1, 2021. Diagnosis of RDS, gestational age of 28–34 weeks and weight ≥ 1 kg were considered as inclusion criteria. Congenital anomalies such as congenital cyanotic heart diseases, digestive system anomalies and chromosome abnormalities were the exclusion criteria.RDS diagnosis and its severity was conducted based on classic clinical presentation of RDS includes grunting respirations, retractions, nasal flaring, cyanosis, and increased oxygen requirement, together with diagnostic finding and onset of symptoms shortly after birth .Sample size was calculated at 95% confidence interval (CI) and 80% power based on a similar study  with staA random number sequence was generated by a person who was not involved in the study, using computer random sequence Stata software version 12. Allocation concealment was performed using numbered, opaque, and sealed envelopes that were opened by the primary researcher at the randomization time. Therefore, all participants had equal chances of being assigned to any group.The neonates, data collectors, assessors, and analysts were unaware of the type of surfactant in each group and were blinded, making this study triple-blind.Two types of surfactants were used for intervention in this study. One group received BLES 135 mg/kg  and the other group received Survanta (Beraksurf®) 100 mg/kg . Drug administration in both groups was performed intratracheally for 10 min. The first dose was given immediately after hospitalization, and if it was not required at that time, it was prescribed in the hospitalization period and with deterioration of RDS (this deterioration was not justifiable with other causes).Chest radiography was performed in both groups 6 h after the first dose. Based on radiographic findings and neonate clinical condition, second and third dose were either prescribed or unfixed.The number of days required for the use of invasive mechanical ventilation (ventilator) and non-invasive continuous positive airway pressure (CPAP) was recorded for all neonates. Some complications were evaluated. These complications are PDA (assessed by echocardiography), IVH (assessed by sonography), sepsis (using blood culture), and pneumothorax  and pulmonary hemorrhage . The mortalities in both groups were also recorded.P value < 0.05 was considered as statistically significant. The analysis was performed in two descriptive and inferential sections. Descriptive, quantitative and categorical data were presented as mean ± standard deviation (minimum–maximum) and frequency (percentage), respectively. Based on quantitative data of comparison between the 2 groups, analyses were performed using independent sample t-tests. Additionally, analyses based on categorical data between the 2 groups were done using Chi-square test. Also, the risk ratio (RR) was calculated for complication and mortality of RDS.Data analysis was performed with Stata software version 12 , and P = 0.0640). The mean of the collective birth weights of neonates in Beraksurf group was 1664.53 ± 631.27 kg and 1625.55 ± 567.86 kg in the BLES group. There was no significant difference between the mean of birth weight in both groups (P = 0.646). Baseline and demographic information of the two groups is demonstrated in Table The mean of the gestational age of neonates in Beraksurf and BLES surfactant groups were 31.64 ± 1.69 weeks and 31.03 ± 1.96 weeks and the difference was not significant (P = 0.689). In the Beraksurf group, 83.9% (n = 52) of neonates received one dose and 16.1% (n = 10) received two doses of surfactant, while in BLES group, 79.7% (n = 51) of neonates received one dose, 18.8% (n = 12) received two doses, and 1.6% (n = 1) received three doses of surfactant. There was no significant difference in the frequencies of surfactant administration between the two groups (P = 0.560).The mean time to receive surfactant was 4.49 ± 1.18 h in Beraksurf group and 4.24 ± 1.09 in BLES group. Time of receipt of surfactant in Beraksurf group was ≤ 12 h in 35 (54.7%), 2–12 h in 25 (39.1%) and ≥ 12 h in 4 (6.2%) neonates. In BLES group, it was ≤ 12 h in 35 (54.7%), 2–12 h in 27 (42.2%), and ≥ 12 h in 2 (3.1%) neonates. There was no significant difference between the mean post-natal age of surfactant administration in both groups (P = 0.641). Neonates that received Beraksurf spent a mean of 4.39 ± 0.74 days with ventilation support and those that received BLES ventilated for a mean of 4.46 ± 0.64 days, but the difference was not significant (P = 0.937). Also, the mean of days required for a CPAP in Beraksurf group was 4.79 ± 0.48 days which was higher than that of neonates in BLES group at 4.1 ± 0.49 days. This difference was also not significant (P = 0.325).The mean days of hospital stay were 17.66 ± 1.46 days and 16.68 ± 1.46 days in Beraksurf and BLES groups respectively; however, this difference was not significant (P = 0.025) and pulmonary hemorrhage (P = 0.015) in the two groups were statistically significant. The incidence risk of all complications in Beraksurf group was lower than BLES group so that the incidence risk of pneumothorax and pulmonary hemorrhage were 0.35 (95% CI: 0.13–0.93) and 0.33 (95% CI: 0.12–0.86) in Beraksurf group compared with BLES group. Other risk differences were not significant.Table The prevalence of RDS is 45% , and witP value of its difference was borderline. Compared with BLES group, the RR estimates of pneumothorax and pulmonary hemorrhage were 0.35 and 0.33 in Beraksurf group and were significant. Also, the incidence risk of other complications in Beraksurf group was lower than BLES group and were not significant. There was no significant difference between mean of hospital stay, ventilator, and CPAP days in the two groups.In this study, we compared a new and cost-effective surfactant produced in Iran with a relatively more expensive and inaccessible surfactant. Based on our results, there were no significant differences between frequencies of gender, type of delivery and diabetes in mothers in BLES and Beraksurf groups. In the present study, neonates treated with Beraksurf experienced the complications such as PDA, IVH, sepsis, pneumothorax, and pulmonary hemorrhage less frequently than those treated with BLES. From all these assessed complications, only pneumothorax and pulmonary hemorrhage caused significant statistical difference between the two groups. Also, the mortality rate in Beraksurf group was 17.2% and was lower than BLES group (31.3%), however, the The Beraksurf treatment in our study is a cost-effective and available surfactant; based on our results, its effect on RDS complications is similar to BLES. When compared with BLES, incidence risk of some complications such as pneumothorax and pulmonary hemorrhage in RDS patients is lower with this surfactant. BLES was first used in 1983; this Canadian surfactant is a powerful and effective drug in RDS treatment and is a fully natural human surfactant produced from amniotic fluid or biosynthetic materials. But, the Survanta is a natural corrected surfactant that is produced in some countries. In Iran this surfactant with the brand name Beraksurf® was produced by Tekzima pharmaceutical company. The chemical component of Beraksurf is similar to other Survanta and it is expected to be an effective surfactant with lower price and more availability.Some studies compared the non-Iranian Survanta with other surfactants (given the similarity of Iranian Survanta to Survanta manufactured in other countries). In a study conducted by Macooie et al. , the effConsidering the effectiveness of neonatal RDS treatment and ventilator-induced complications in these patients, we recommend the use of surfactant replacement therapy. Our scientific study assessed Beraksurf®, an Iranian Survanta, as effective as international surfactant products on neonatal RDS treatment and safe enough to be prescribed. Hence, we suggest the clinicians to consider it as an alternative when international brands are more expensive and inaccessible.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Jeongeupia species, which have been screened, isolated, and biochemically characterized from chitin‐amended soil samples. Through metabolic characterization, whole‐genome alignments, and phylogenetic analysis, we could demonstrate how the investigated strains differ from the taxonomically closest strain Jeongeupia naejangsanensis BIO‐TAS4‐2T (DSM 24253). In silico analysis and sequence alignment revealed a multitude of highly conserved chitinolytic enzymes in the investigated Jeongeupia genomes. Based on these results, we suggest that the two strains represent a novel species within the genus of Jeongeupia, which may be useful for environmentally friendly N‐acetylglucosamine production from crustacean shell or fungal biomass waste or as a crop protection agent.Chitin is the second most abundant polysaccharide worldwide as part of arthropods' exoskeletons and fungal cell walls. Low concentrations in soils and sediments indicate rapid decomposition through chitinolytic organisms in terrestrial and aquatic ecosystems. The enacting enzymes, so‐called chitinases, and their products, chitooligosaccharides, exhibit promising characteristics with applications ranging from crop protection to cosmetics, medical, textile, and wastewater industries. Exploring novel chitinolytic organisms is crucial to expand the enzymatical toolkit for biotechnological chitin utilization and to deepen our understanding of diverse catalytic mechanisms. In this study, we present two long‐read sequencing‐based genomes of highly similar  Jeongeupia species “wiesaeckerbachi” was isolated from soil samples, characterized biochemically, and sequenced with the long‐read platform PacBio Sequel IIe. In silico analysis unraveled genomic differences to the closest related type strain Jeongeupia naejangsanensis TAS4‐2 in addition to an usually extensive chitinolytic machinery.In this study, a novel chitinolytic  Parallelly to cellulose, which differs structurally through a lack of an amido‐functionality, and imparts stability and structure to higher plants, chitin is the principal structural component of fungal cell walls and cuticles of insects and crustacean exoskeletons, algal cell walls, and mollusks endoskeletons in aquatic organisms ‐linked Rinaudo, .Proteobacteria, Bacteroides, Actinobacteria, and Firmicutes  and corresponding deacetylated derivatives comprise the food‐, cosmetic‐, wastewater treatment‐, and medical industries technologies, extensive research on chitinases has been conducted  gene analysis  MagBead Kit from Zymo Research and HMW genomic DNA (gDNA) shearing was conducted with g‐TUBEs (Covaris) according to the manufacturer's manual.2.22O in a 5 L glass beaker and vigorously stirred, rapidly swelling to white colloidal chitin. The solution was incubated overnight at 4°C without stirring and neutralized the following day by adding excessive amounts of deionized water and subsequent centrifugation in a Beckman JLA8.1000 rotor for 15 min at 10,000g until pH 5 of the supernatant was reached. CC was harvested, autoclaved, and kept in the fridge until utilization in liquid chitinase screening media (CSM) or agar plates. The recipe was adapted and modified from , adjust to pH 6.5 for plates or 7 for liquid medium. After autoclaving, 0.001 g/L ZnSO4 and MnCl2 were added from sterile filtrated stock solutions before pouring of agar plates/inoculation of liquid media.Colloidal chitin (CC) was prepared according to  and covered with tin foil. After incubation at room temperature for 2 weeks, portions of the amended soil samples were transferred to sterile 50 mL falcon tubes and filled to 50 mL with sterile 1X PBS. Soil samples were incubated in a thermal shaker at 30°C and 600 rpm for 30 min. Supernatants were streaked out on CSM agar plates with different pH  using inoculation loops and incubated at 28°C for 2–3 days. Colony‐forming units (CFU) surrounded by halos were streaked onto separate CSM agar plates of the respective pH until axenic strains were obtained  primer pair F27‐5′‐AGA GTT TGA TCC TGG CTC AG‐3′ and 1492R‐5′‐AAG GAG GTG ATC CAA GCC‐3′ at 55°C annealing temperature. After deploying the DreamTaq DNA polymerase (Thermo Fisher Scientific), the length and quantity of PCR products were validated via agarose gel electrophoresis, gel bands were excised, and gene fragments extracted with the Monarch DNA Gel Extraction Kit (New England BioLabs GmbH) according to the protocol. Eurofins Genomics Europe Sequencing GmbH conducted Sanger sequencing with the provided primer pair F27 and 1492R. Finally, the Geneious Prime software (v.2022.0.1) was used for read quality control, alignment, and assembly to obtain near full‐length rRNA sequences, which were compared to NCBI's 16S rRNA gene database through their BLASTn suite  and incubated in 20 mL Tryptic Soy Broth medium in 150 mL baffled shaking flasks at 120 rpm and 28°C overnight. HMW gDNA was extracted according to the instructions of the Quick‐DNA HMW MagBead Kit (Zymo Research).To assess the quantity and purity of the obtained DNA, 260/280 nm absorption ratios and concentrations were measured with a photometer  and a Qubit 4 fluorometer with the Qubit 1X dsDNA HS Assay‐Kit (Thermo Fisher Scientific). To confirm the high molarity of the gDNA, fragment sizes were analyzed with a Femto Pulse capillary electrophoresis instrument (Agilent Technologies).g in a tabletop centrifuge. This yielded DNA fragments with a size of ca. 12 kbp, as confirmed with Femto Pulse. Subsequently, HiFi libraries were prepared according to the SMRTbell prep kit 3.0 manual, fusing barcoded adapters to the samples (Pacific Biosciences). Libraries were stored at −20°C until the day of sequencing, where primers and the polymerase bound the samples with the Sequel II Binding Kit 3.2 (Pacific Biosciences), closely following the manufacturer's recommendations.When samples passed the quality control, shearing of 8 µg gDNA in 150 µL Elution Buffer was conducted with g‐TUBEs (Covaris), utilizing 17002.5.2Whole genome sequencing was performed on a Sequel IIe platform (Pacific Biosciences) on a single SMRT cell (lot number 418096) with the following parameters: 2 h of pre‐extension, 2 h of adaptive loading (target p1 + p2 = 0.95) for a final on‐plate concentration of 85 pM and a 30‐h long movie window for signal detection  and the ‐pacbio parameter was deployed; otherwise, standard settings were utilized. Log files can be found in Supporting Information: Data. Annotation was performed utilizing NCBI's PGAP  to separate the barcoded reads, obtained FASTQ raw read files were assembled utilizing the Canu assembler 2.0  were retrieved with dbCAN 3.0  , respectively, chitinolytic microorganisms could putatively be enriched, as previously reported  and 98.01% (J. sp.), respectively, Jeongeupia chitinilytica's 16S rRNA gene showed the second most sequence homology. The transitory names were awarded based on these results, indicating that the investigated organism is J. naejangsanensis, leading to “J. n.” for the first strain. Due to visible morphological differences on the screening plates earlier in the study, possibly originating from the presence of a contaminant, the second candidate strain was thought to be a deviant Jeongeupia. This hypothesis was later reinforced by an aligned nucleotide identity‐based taxonomic analysis, leading to the name “J. sp.” When deploying the 16S rRNA gene sequences extracted from the novel high‐quality genomes (gene IDs pgaptmp_00343 [J. n.] and pgaptmp_1503 [J. sp]) to a BLASTn query, the colony 16S rRNA gene PCR‐based results could be confirmed with percent identities of 99.06%, E values of 0.0 and query coverages of 97%, respectively.Both candidates were identified as 3.1.1J. naejangsanensis BIO‐TAS4.2T  and compared to the taxonomically closest strain 2T Table . As exped‐lyxose, l‐arabitol, and capric acid, all of which the type strain can utilize as a carbon source , J. naejangsanensis , J. sp. HS‐3 (RefSeq GCF_015140455.1), and J. sp. USM3 (RefSeq GCF_001808185.1).Barcode adapter fused genome libraries were sequenced along other biosamples with PacBio's long‐read platform Sequel IIe (Pacific Biosciences). Subsequently, reads were demultiplexed (binned according to the barcode) computationally. The overall HiFi reads from the circular consensus sequencing mode were satisfactory in quantity and quality, with a Q36 score, translating into a 99% accurate base calling. Owed to the delicate balancing act regarding library concentrations during multiplexing, the ry Table . Throughry Table , reads wNeisseriales database  as the closest available reference . The lack of fragmented BUSCOs as opposed to the reference strain could originate in the gapless assembly enabled by the long‐read sequencing platform. Duplicated orthologues, as prevalently seen in the J. sp. genome, stem exclusively from its minor secondary and tertiary contigs, which were not omitted as contaminations by the Canu 2.0 assembler.To further evaluate the assembled genome qualities, BUSCO 5.3.2 guided assessment of orthologue gene set completeness was performed, utilizing the order of the y Figure , on par 3.2.1Average nucleotide identity (ANI)‐based identification: The ANI values, widely accepted as a computational tool to define species boundaries and confirm identities of Bacteria and Archaea, were routinely assessed by the annotation pipeline PGAP , the highest respective ANI values were 87.61% for both J.n. and J. sp. compared to J. naejangsanensis BIO‐TAS4‐2T . In the case of submitting the genus Jeongeupia as a reference, the best hit changed to an aligned nucleotide identity of 85.9% for J. sp. with the species J. chitinilytica , instead . Furthermore, it is reported for several genera, for example, Variovorax or Stenotrophomonas, to be defined more loosely by the ANIs, with lower cutoff values of 88% and 88.5%, respectively  and the type strain BIO‐TAS4‐2Horizontal gene transfer and adaptation to local habitats, driven by interactions between local bacterial communities  with a confidence value of 97% and a delta statistic of 0.26 , and the central auxiliary enzyme of family 10 (AA10), the lytic polysaccharide monooxygenase (LPMO), were of interest , type strain J. naejangsanensis BIO‐TAS4‐2 exhibited 21 potentially chitinolytic enzymes, with 13 GH18, 3 GH19, 2 GH20, a single LPMO and one, as partial chitinase annotated putative protein   ; data curation ; formal analysis ; investigation ; methodology ; software ; validation ; visualization ; writing—original draft . Daniel Garbe: Conceptualization ; supervision (lead); writing—review and editing . Thomas B. Brück: Conceptualization ; funding acquisition ; project administration ; resources ; supervision ; writing—review and editing .The authors declare no conflict of interest.None required.