Journal of Vision, 17(3), 10, One of the most precise methods to establish psychometric functions and estimate threshold and slope parameters is the constant stimuli procedure. The large distribution of predetermined stimulus values presented to observers enables the psychometric functions to be fully developed, but makes this procedure time-consuming. Adaptive procedures enable reliable threshold estimation while reducing the number of trials by concentrating stimulus presentations around observers’ supposed threshold. Here, the stimulus value for the next trial depends on observer’s responses to the previous trials. One recent improvement of these procedures is to also estimate the slope (related to discrimination sensitivity). The Bayesian QUEST+ procedure (Watson The online version contains supplementary material available at 10.3758/s13428-022-02001-4. The purpose of psychophysics is to accurately and objectively evaluate the relationship between physical dimensions of stimuli and perceptual responses. During psychophysical procedures, stimuli varying along a given physical dimension are presented to observers who have to indicate whether they perceive the stimuli (detection), or whether they differ from a reference stimulus (discrimination).One of the most straightforward psychophysical methods is the method of constant stimuli (CS). In this procedure, the distribution of stimulus values covers a large range of values and is specified before the experiment. The advantage of this method is that the psychometric function  can be fully developed. Thresholds  and slopes  can thus be determined accurately. Nevertheless, there are also many trials that are far from the observers’ threshold and therefore non-informative. This makes the CS procedure time-consuming and requires patience from both observers and experimenters.Adaptive procedures have been designed and developed to increase measurement efficiency, i.e., to obtain valid, precise, and reliable threshold estimation while minimizing experimental time and observers’ efforts. They do not require responses to a complete set of physical stimulus values, but concentrate stimulus presentations around observers’ threshold. We will not describe these procedures extensively; the reader is referred to the excellent reviews by Treutwein  and LeekAmong adaptive procedures, nonparametric methods, called staircase procedures, use responses to the previous trial or a sequence of trials, within an adaptive track, to select the stimulus physical value for the next trial. No assumption is made about the shape of the psychometric function, except that it is strictly monotonic. Stimulus placement rules along the array, step sizes, and termination rules differ according to the specific method used. For instance, in a simple up-down staircase procedure, the stimulus value is changed by a fixed step size after each response. Stimulus value adjustments will remain in the same direction unless the observer changes response category   parameters of the psychometric function are estimated, typically with a maximum-likelihood fitting algorithm. The new parameter estimates, which are refined over the course of the experiment, are used to determine the most informative stimulus to present next. A Bayesian variant of these procedures, the QUEST procedure  of the psychometric function .r = .60) as that in Farahbakhsh et al.  that began at stimulus levels far above, far below, or at the supposed threshold . Each track was stopped after 15 response reversals or 10 presentations of the maximum or minimum stimulus value. The whole staircase procedure could continue up to 150 trials. At the end of the procedure, a mean threshold value was classically computed based on the last five reversals for each track. Then, the 50% threshold estimation was computed as the mean of the three tracks’ threshold mean values. By developing psychometric functions, we were able to obtain second threshold estimations, as well as slope estimations.Pyes) as a function of the stimulus level (x). A “yes” response is expected for the highest stimulus values, and its probability increases with the increase in stimulus values. This function has been implemented in Brainard’s code as follows:The QUEST+ procedure was programmed using a free extension for MATLAB, made available in 2017 by Brainard on his GitHub repository, which takes up Watson’s implementation on Mathematica in his 2017 article. The underlying function was a cumulative normal distribution. Such a model describes the probability of responding “yes” . The standard MATLAB function erf(z) returns the error function evaluated for each element of z. This MATLAB implementation is equivalent to the function proposed for Mathematica by Watson  or from our pilot experiments. We decided to use uniform distributions of stimulus and parameter domains , meaning that we did not introduce any explicit Bayesian priors—although we made assumptions about the models and the domain boundaries. This way, we avoided the additional risk of measurement biases or experimenter errors /2, with x.75 and x.25 corresponding to the stimulus values at which the probability of a given response  is equal to 75% and 25%, respectively. The JND is a measure of the spread of the psychometric function and thus is inversely related to the slope. Because JND distributions were moderately asymmetric and skewed, we transformed our JND data for analyses. We chose a logarithmic transformation based on the Box–Cox procedure  and just-noticeable differences (JNDs). The PSE is the point at which a comparison stimulus is judged equal to a reference stimulus , and gives information about possible response biases (the degree to which “yes” or “no” responses are preferred) or systematic errors. The JND is the smallest detectable difference between two stimuli, and was calculated as follows: lme4, Bates et al., F tests on fixed effects, we used Satterthwaite’s approximation  for each model against the null model .For the main analyses, linear mixed models .To quantify the degree to which the three procedures are related, we calculated Pearson or Spearman pairwise correlations . Because correlations only measure the strength of a linear relationship between two variables, we conducted Bland–Altman analyses . As random effects we had intercepts for participants. To examine if a response bias existed, we also compared mean PSE values obtained for each block (or procedure) to the value of the reference stimulus, using Bayesian one-sample The first experiment aimed at comparing PSEs and JNDs obtained with the three procedures in a typical psychophysical task: the size judgment of a disk presented in central vision.M = 23.8; SD = 6.5) contributed data in this experiment (three of them were the authors). An additional four adults were tested but excluded because of failure to complete the experiment (1) or because the standard deviation of the psychometric function estimated by the QUEST+ algorithm reached the ceiling (3). It is thus likely that their JNDs do not reflect their true perceptual sensitivity. In addition, for two of these participants, the adaptive tracks of the staircase procedure did not converge.Twenty-two participants  aged 18 to 41 years .2 luminance gray background. Disk contours were homogeneously blurred by a cumulative Gaussian gradient covering 20% of the diameter , and lapse values between 0 and 0.06 (0.01 dva step). Fig. , we used the Sn method .MQ+ = 1.58 dva, SDQ+ = 0.07, rangeQ+: 1.47–1.69; MCS = 1.57 dva, SDCS = 0.07, rangeCS: 1.36–1.67; MStaircaise = 1.56 dva, SDStaircase = 0.09, rangeStaircase: 1.41–1.73), LMM: F = 1.46, p = .24, and BF01 = 2.8. A Bayesian one-sample t-test revealed anecdotal evidence for bias when PSEs were measured using the CS, BF01 = 0.87, and staircase procedures, BF01 = 0.58, but not for the QUEST+ procedure, BF01 = 1.4.Figure r(19) ≥ .79 and all ps < .001. The strongest correlation, r(19) = .86, p < .001, was observed for the two adaptive procedures  = 5.45, p = .008; marginal R2 = .083. Tukey’s honestly significant difference (HSD) post hoc analyses  in comparison with the CS procedure , p = .008. QUEST+ and CS did not differ from the staircase procedure , p = .59 and p = .088, respectively.JNDs were slightly affected by the procedure, as illustrated in Fig. r(19) = .75, p < .001  = 2.30, p =.11, and BF01 = 1.62, nor on JNDs, LMM: F = 0.28, p =.76, and BF01 = 6.34; they remained constant through the entire experiment . We did not observe any block effect on PSEs, LMM: see Fig. .Fig. 7MeIn this experiment we compared PSE and JND estimates obtained from the QUEST+, CS, and staircase procedures for feature orientation judgments, another low-level physical dimension. We increased task difficulty by presenting stimuli in peripheral vision.M = 22.7, SD = 6.2) contributed data in this experiment (three of them were the authors). An additional 11 participants were tested but excluded for failure to calibrate (1), because the final standard deviation estimated by the QUEST+ algorithm reached the maximum ceiling (3), because they did not perform the task correctly, yielding aberrant values for at least one procedure (4), or for a combination of the two latter points (3).Twenty-three participants  aged 18 to 42 years  by a digital micromirror video projector . The room was lit only by the projected image. Participants sat ~140 cm from the screen, with their head stabilized by a chin and forehead rest. We used a video-based eye tracker  to control for central fixation during peripheral stimulus presentation.2 luminance gray background. The reference gabor was tilted 20° to the right whereas the test gabor tilt angles could vary from 5° to 35° to the right. A 0.7 × 0.7 dva black cross (2 pixels line width) served as a fixation point. At the beginning of each psychometric procedure, and for each participant, a different visual noise mask was generated. The masks consisted of 5 × 5 dva squares composed of gray-level pixels whose values were randomly picked between 0 and 100 (0 being black and 255 white).Stimuli, two 2 × 2 dva black and white gabors , were displayed on a 7.5 cd/mA trial always started with the presentation of a fixation cross displayed at the center of the screen Fig. , frame 1In the CS procedure, the set of tilt angle differences between the test and reference gabors consisted of seven possible values from −9° to 9°  spaced by 3°. Each tilt angle difference was randomly presented 24 times during the session. Two ±15° tilt angle differences were also inserted eight times each throughout the course of the session.In the staircase procedure, the initial tilt angle differences were −28°, 0°, and 28°, depending on the simple up-down track considered. Then the boundaries were set to ±24°. In each track, the angle difference changed by 4° after the first two response reversals and by 2° for the following reversals.In the QUEST+ procedure, the set of tilt angle differences consisted of all the possible values from −24° to 24° linearly spaced by 2°. The possible values for the mean were the same as for the stimulus domain. Standard deviation could take values between 1 and 20 (step of 0.1), and lapse, values between 0 and 0.08 (step of 0.01°).Sn method, with a criterion of 3). An additional analysis with all participants showed that the exclusion of these outliers did not change the statistical conclusions on PSEs and JNDs, but that some correlations between estimates were artificially increased .F = 3.08, p =.057, even if evidence speaking in favor of this null hypothesis is anecdotal, BF01 = 1.23. For the QUEST+, CS, and staircase procedures, the mean PSEs were 1.31° , 0.94°  and −0.15° , respectively. Participants were accurate in all procedures, as the mean PSE values never differed from the critical value of zero  = .69, p < .001. The other two coefficients were similar although slightly smaller, both r(18) = .45, p = .045 and .046  = 5.98, p = .005, marginal R2 = .092. Tukey HSD post hoc analyses showed that the QUEST+ procedure yielded the highest measures of sensitivity  in comparison with both the CS procedure  and the staircase procedure , which did not differ from each other (p = .84).Contrary to PSEs, the procedure significantly impacted the JNDs see Fig. , Fr(18) = .62, p = .005. Whereas a comparable correlation was obtained for the CS and staircase procedures, r(18) = 0.59, p = .007, it was lower and failed to reach significance for the two adaptive procedures, r(18) = 0.42, p = .065  = 0.62, p = .54, and the Bayesian ANOVA analysis, BF01 = 4.86.Figure F = 13.6, p < .001; marginal R2 = 0.16. Tukey HSD post hoc analyses showed that JNDs significantly decreased between the first and third blocks (p < .001) and the second and third blocks (p = .029). A similar trend can be seen between the first and second blocks but failed to reach significance (p = .052).A learning effect however occurred for sensitivity, LMM: In this experiment we compared the two estimates obtained from the QUEST+, CS, and staircase procedures for a higher-level dimension: temporality, assessed for stimuli presented in the periphery. This experiment differed from the preceding ones in that it consisted of the presentation of a single magnitude (in terms of duration) per trial. Participants were asked to perform a temporal-order judgment (TOJ) task, i.e., to report which of two stimuli appeared first. We measured points of subjective simultaneity (PSS), which corresponded to the delay with which one stimulus had to be presented before the other for observers to perceive them as appearing at the same time. The JND represents the smallest interval at which the participants could reliably determine which stimulus was presented first.M = 22.9, SD = 5.9) contributed data in this experiment . An additional four participants were tested but excluded for failure to perform the task (3) and because they mistakenly pressed the wrong buttons (1).Twenty-four participants  aged 18 to 41 years . One gabor was vertical and the other horizontal. A 1 × 1 dva black cross (2 pixels line width) served as a fixation point.The gray background had a luminance of 20 cd/mA trial always started with the presentation of a fixation cross displayed at the center of the screen Fig. , frame 1In the CS procedure, the set of SOAs consisted of all the possible values from 3 to 18 frames (one frame = 4.2 ms), spaced by three frames. Each SOA was randomly presented 24 times during the session. Three other SOAs of 0, 24, and 30 frames were also inserted 12 times each throughout the course of the session, for a total of 180 trials. In half of the trials, the vertical gabor was presented first, while in the other half, the horizontal gabor appeared first. In the latter case, SOAs were multiplied by −1. A null SOA corresponded to simultaneity. The positions of the gabors were counterbalanced across SOAs.In the staircase procedure, the initial SOAs were −40, 0 and 40 frames, depending on the simple up-down track considered. In each track, after the first response reversal, the SOA changed by four frames. After the second reversal, it changed by three frames, and by two frames for the following reversals.In the QUEST+ procedure, the set of SOAs consisted of all the possible values from −60 to 60 frames, linearly spaced by 1. The possible values for the mean were all the possible values spaced by one frame from −40 to 40 frames. Standard deviation could take values between 1 and 50 (step of 1) and lapse values between 0 and 0.08 (step of 0.01).In the two adaptive procedures, the first gabor randomly appeared in one of the two positions.Sn method, with a criterion of 3). As in the previous experiment, an additional analysis with all participants showed that the exclusion of these outliers did not change the statistical conclusions on PSEs and JNDs, but that some correlations between estimates were artificially increased  in the staircase procedure, .F = 0.62, p = .54, and BF01 = 4.90 , but not with the staircase one .As in the previous experiments, thresholds (PSSs here) did not vary as a function of the procedure, see Fig. . There wr(19) = .46, p = .036. The correlation between PSSs for the two adaptive procedures and for the CS and QUEST+ procedures failed to reach significance, r(19) = .42, p = .057 and r(19) = .25, p = .27, respectively . There was no bias for one procedure over another .The three Bland–Altman plots Fig.  show thaF = 7.45, p = .002, marginal R2 = 0.15  in comparison with the CS procedure , p = .001. JNDs from the staircase procedure, however, did not differ significantly  from those measured using the QUEST+ (p = .12) or the CS (p = .20) procedures.Unlike PSSs, sensitivity was impacted by the procedure, see Fig. . As indir(19) = .46, p = .035. The correlations between JNDs for the two adaptive procedures and for the CS and QUEST+ procedures did not reach significance, r(19) = −.14, p = .055 and r(19) = .40, p = .073, respectively.As for PSSs, the only significant correlation between JNDs was observed for the CS and staircase procedures, a = −1) and C (a = 0.85) differed significantly from zero , showing that bias increased when sensitivity decreased. The two corresponding agreement intervals were comparable, 14 and 13 frames.Again, the strongest degree of agreement was observed for the two adaptive procedures  = 1.22, p = .30, and BF01 = 3.2.First, we examined whether PSSs and JNDs improved throughout the experiment, as a result of a possible learning effect Fig. . LMM conF = 3.85, p = .029, marginal R2 = .086. Tukey HSD post hoc analyses revealed that there was only a trend for JNDs to be smaller in the first block of trials  in comparison with both the second  and third blocks , which did not differ from each other (p = .10).An improvement was however visible in terms of sensitivity: JNDs significantly decreased over the different blocks of trials, BF01= 0.15 and 0.69, Bayesian one-sample t-test). This response bias disappeared only on the last block of trials (BF01 = 3.16). Overall, mean PSS values decreased from −1.21 (SD = 1.86) to −0.45 (SD = 2.39).Further analyses indicated that our participants showed a response bias; their mean PSS values differed from the zero value on the first two blocks  estimates in the same range as estimates obtained with two other psychophysical procedures commonly used in many laboratories, namely, the CS and the nonparametric staircase procedures. Overall, as expected, we observed important correlations between the PSEs of the three procedures. This study thus provides robust empirical evidence that the QUEST+ procedure, which requires a small number of trials, constitutes a reliable and efficient tool to measure perceptual thresholds and detect perceptual biases. This result is extremely important for studies interested in specific populations . Notably, some authors combined elements of SDT and Bayesian adaptive inference to determine such threshold estimates that are free of decision criterion confounds , considered as the reference frame for assessing sensitivity and bias in perceptual decision-making, provides a definition of the threshold that is independent of both the psychophysical method used and the context of the task  and/or by using appropriate priors. QUEST+ has proved to be generalizable to different stimulus dimensions involved in perception: size, orientation, and temporality. These findings motivate us to use this Bayesian method in other psychophysical tasks and sensory modalities.ESM 1(DOCX 28 kb)


Previous studies have reported the role of circular RNAs (circRNAs) in the progression of non‐small‐cell lung cancer (NSCLC). SWT1‐derived circRNAs were confirmed to affect the apoptosis of cardiomyocytes; however, the biological functions of SWT1‐derived circRNAs in cancers are still unknown. Here, we investigated the potential role of SWT1‐derived circRNAs in NSCLC.We used quantitative real‐time polymerase chain reaction (qRT‐PCR) to measure the expression of circSWT1 in NSCLC tissues and paired normal tissues. The potential functions of circSWT1 in tumor progression were assessed by CCK‐8, colony formation, wound healing, and matrigel transwell assays in vitro and by xenograft tumor models in vivo. Next, epithelial‐mesenchymal transition (EMT) was evaluated by western blotting, immunofluorescence, and immunohistochemistry (IHC). Moreover, circRIP, RNA pulldown assays, luciferase reporter gene assays, and FISH were conducted to illuminate the molecular mechanisms of circSWT1 via the miR‐370‐3p/SNAIL signal pathway. Then, we knocked out SNAIL in A549 and H1299 cells to identify the roles of circSWT1 in the progression and EMT of NSCLC through SNAIL. Finally, circSWT1 functions were confirmed in vivo using xenograft tumor models.CircSWT1 expression was significantly upregulated in NSCLC tissues, and high expression of circSWT1 predicted poor prognosis in NSCLC via survival analysis. In addition, overexpression of circSWT1 promoted the invasion and migration of NSCLC cells. Subsequently, we found that overexpression of circSWT1 induced EMT and that knockdown of circSWT1 inhibited EMT in NSCLC cells. Mechanistically, circSWT1 relieved the inhibition of downstream SNAIL by sponging miR‐370‐3p. Moreover, we found that these effects could be reversed by knocking out SNAIL. Finally, we verified that circSWT1 promoted NSCLC progression and EMT in xenograft tumor models.CircSWT1 promoted the invasion, migration, and EMT of NSCLC. CircSWT1 could serve as a potential biomarker and a potential therapeutic target for NSCLC. CircSWT1 expression was significantly upregulated in NSCLC tissues, and high expression of circSWT1 predicted poor prognosis in NSCLC via survival analysis. CircSWT1 can act as a sponge of miR‐370‐3p. Overexpression of circSWT1 induced NSCLC progression by inducing cell EMT via miR‐370‐3P/Snail axis. The majority of instances of lung cancer (85%) are non‐small‐cell lung cancer (NSCLC), which is the most prevalent pathological type. With smoking cessation and improvements in early diagnosis and treatment, the death rate from lung cancer has steadily declined, and survival rates have been rapidly improving, particularly for NSCLC. All cancer patients have a 67% five‐year survival rate on average. However, patients with lung cancer have a 21% five‐year survival rate, one of the lowest among all cancer patients. Additionally, the greatest number of deaths are still from lung cancer—approximately one‐quarter of all cancer deaths are due to lung cancer.Circular RNA (circRNA), a vital species of endogenous noncoding RNAs, is a form of single‐stranded RNA that mostly consists of one or more exons and has been found to be crucial in the control of transcriptional and post‐transcriptional gene activity.In the process known as epithelial‐mesenchymal transition (EMT), epithelial cells lose their unique traits and acquire those of mesenchymal cells. Growing evidence has inked EMT to many tumor functions, including tumor migration, intravasation, and resistance to therapy.CircRNAs have a significant role in the growth of many tumor types.According to earlier research, circRNAs have been linked to the progression of many different types of solid tumors. However, circSWT1 has gotten very little attention. In this study, we found that circSWT1 level was significantly upregulated among SWT1‐derived circRNAs in NSCLC, and a high level of circSWT1 predicted poor progression of NSCLC patients. Moreover, in vitro and in vivo experiments revealed that circSWT1 facilitated invasion, metastasis, and EMT of NSCLC cells. Mechanistically, we also found that circSWT1 sponged miR‐370‐3p and promoted downstream SNAIL expression to hinder tumor progression and EMT in NSCLC. However, this phenomenon was reversed when the SNAIL was knocked out. Our study confirmed that circSWT1 could promote NSCLC invasion, migration, and EMT via the miR‐370‐3p/SNAIL axis. As a result, circSWT1 may be used as a potential biomarker and therapeutic target for NSCLC.22.1Ninety‐six NSCLC specimens and matched normal tissues were obtained from the Second Affiliated Hospital of Nanchang University from 2014 to 2015. After surgical excision, pathologists categorized all of the tissues, which were then all preserved at −80°C and utilized to create a tissue microarray (TMA) for immunohistochemistry (IHC) staining. Each patient gave their informed permission before surgery, and the study was given the go ahead by the Second Affiliated Hospital of Nanchang University's ethics committee.2.2The human normal bronchial epithelial cells (HBE) and human NSCLC cell lines  were purchased from the Cell Bank of the Chinese Academy of Sciences . In DMEM and RPMI‐1640 with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin, all the cells were grown (Yeasen). These cells were raised at 37 °C with 5% CO2 in a cell incubator (Thermo Fisher Scientific). Following the manufacturer's instructions, lentiviral vectors carrying circSWT1 and shcircSWT1 were bought from Genomeditech and transfected into A549 and H1299 cells. Genomeditech supplied the CRISPR Cas9‐gDNA technology that was utilized to eradicate the SNAIL gene from the A549 and H1299 cell lines. The sequences of shcircSWT1 are reported in Additional file 1: Table 2.3All these assays were described in our previous study.Assays for colony formation were carried out to assess the viability of NSCLC cells. In a word, 1000 cells were added to 6‐well plates and incubated for 2 weeks. After that, we counted the colony numbers after staining.2. Finally, the cells were counted after staining by 0.1% crystal violet (Sigma) via a light microscope.Matrigel transwell assays were used to measure cell invasion. NSCLC cells were first suspended in serum‐free media before being put on top of chambers that had been coated with 1 μg/μl Matrigel (BD Biosciences). The bottom of the chambers was filled with a medium containing 10% FBS. The cells were then fixed with 4% paraformaldehyde after being cultured for 48 hours at 37°C and 5% COWound‐healing experiments were used to evaluate cell migration. In brief, NSCLC cells were incubated in six‐well plates until 95% confluence was obtained. Thus, we created a vertical scratch in the center of the six‐well plates and filled them with serum‐free media. Finally, migration areas were imaged and measured every 24 hours by Image J.2.4These experiments have been carried out in prior studies,2.5A549‐circSWT1, H1299‐shcircSWT1 and control cells were grown on cell slides. Next, the cells were processed by fixation, permeabilization, and blocking with goat serum albumin incubation (Origene). All cells were then treated with particular primary antibodies for 12 hours at 4°C. As a result, the cells were treated for 1 hour at 37°C with a secondary antibody. DAPI (Yeasen) was added to the slides, and cells were photographed using a fluorescence confocal microscope. The antibody is presented in Additional file 4: Table 2.6RNA isolation and qRT‐PCR analysis were carried out in the same manner as in prior studies2.7This experiment was done to further verify the PCR products. The cDNA and gDNA reverse transcription and isolated from H1299 cells were amplified via PCR with convergent and divergent primers. Furthermore, the PCR products were run on an agarose gel. The results were analyzed by a gel imaging system (Tanon). Additional file 5: Table 2.8We established pGL3‐LUC‐circSWT1, pGL3‐LUC‐SNAIL, mutant pGL3‐LUC‐circSWT1, and mutant pGL3‐LUC‐SNAIL for the dual‐luciferase reporter gene experiment. The wild‐type or mutant vectors were then co‐transfected into HEK‐293 T cells with miR‐370‐3p mimics or NC mimics using transfection reagents . After 48 hours of incubation, cells were lysed and centrifuged, and cell supernatants were collected for detection. A dual‐luciferase reporter assay system was used to measure luciferase activity in cell supernatants (Promega).2. Next, cells were washed again and fixed with 1% paraformaldehyde; thus, we dehydrated these cells. The coverslips were hybridized with a specific probe at 37°C overnight. Finally, at room temperature, the coverslips were rinsed and the cell nuclei stained with DAPI. A confocal fluorescence microscope was used to take the pictures . Table For the FISH, cells were cultured on coverslips, immobilized with 4% paraformaldehyde, washed with PBS, and digested by protease K (Sangon) for 5 minutes at 37°C and 5% CO2.9GeneChem (Shanghai) supplied the biotin‐labeled circSWT1 probe for the circRIP assay. In short, after transfection with the biotin‐circSWT1 and NC probes, H1299 cells were fixed in 4% formaldehyde (Sigma‐Aldrich) and then subjected to a number of procedures, including lysis, sonication, and centrifugation. Thus, the supernatant was incubated for 12 hours with M280 streptavidin Dynabeads (Invitrogen). The compound was rinsed in elution buffer and suspended for 2 hours in lysis buffer. TRIzol reagent (Invitrogen) was used to get total RNA from the compound. qRT‐PCR was then used to measure the relative amount of RNA.The Magna RIP kit was used for the RIP assay, which was carried out according to the manufacturer's instructions (Millipore). TRIzol reagent was used to extract total RNA, and qRT‐PCR was utilized to detect the levels of circSWT1, miR‐370‐3p, and circANRIL. Additional file 4: Table 2.10The RNA pulldown experiment was carried out exactly as previously described.2.11Male nude mice aged 5–6 weeks were acquired from Gempharmatech (Nanjing) and raised in a sterile setting in the Center for Experimental Animals of the Second Affiliated Hospital of Nanchang University. All animal tests were carried out in line with the amended Animals (Scientific Procedures) Act 1986 in the United Kingdom and Directive 2010/63/EU in Europe, and were approved by the Ethics Committee of Nanchang University's Second Affiliated Hospital. Subcutaneous injection with A549‐control, A549‐circSWT1, H1299‐control, and H1299‐shcircSWT1 was performed, and the tumors were measured every 3 days. In addition, tumors were collected from the mice after 30 days, made into paraffin sections, and stained with HE, SNAIL, E‐cadherin, N‐cadherin, and vimentin. IHC was used to screen for EMT indicators in these subcutaneous tumors.Furthermore, A549‐circSWT1, H1299‐shcircSWT1, and control cells were injected into mouse tail veins to create pulmonary metastasis tumor models in order to analyze the metastatic capabilities of NSCLC cells affected by circSWT1.2.12t test. To assess variations in the prognosis of patients with NSCLC, the Kaplan–Meier method and the log‐rank test were utilized, and Cox's regression model was used to study independent prognostic factors. The associations between the levels of circSWT1, miR‐370‐3p, and SNAIL were investigated using Spearman correlation analysis. All p values were two‐tailed, with a p value of 0.05 deemed statistically significant.SPSS 23.0 was used to analyze the data (IBM SPSS). The values are displayed as the mean ± standard deviation (SD). The significance of differences between two groups was determined using an unpaired two‐tailed Student's 33.1CircRNA has been linked to the development of a number of malignancies and has been shown to alter patient prognosis, including NSCLC.To further quantify the connection between clinicopathological features and circSWT1 in NSCLC, the level of circSWT1 was measured in 96 NSCLC and matched normal tissues using qRT‐PCR. First, the level of circSWT1 in 96 NSCLC tissues was remarkably upregulated in normal tissues Figure . Further3.2Given the probable relevance of circSWT1 in NSCLC patient prognosis, we conducted a series of studies to evaluate circSWT1's biological roles in NSCLC. qRT‐PCR was used to measure the levels of circSWT1 in HBE cells and 5 NSCLC cells . The results showed that circSWT1 levels were highest in H1299 cells and lowest in A549 cells Figure . As a reMoreover, further studies were conducted to verify whether circSWT1 affects the development of tumors. First, we found that overexpression of circSWT1 could promote the viability of A549 cells and knockdown of circSWT1 inhibited tumor viability, according to CCK‐8 and colony formation assay results Figure . Matrige3.3High group had higher levels of E‐cadherin and lower levels of N‐cadherin than the circSWT1Low group. CircSWT1 levels were shown to be positively correlated with E‐cadherin levels and negatively correlated with N‐cadherin levels . Figure . AccordiNext, the possible binding targets of miR‐370‐3p were then predicted using StarBase 3.0, miRmap, and PITA. The results showed that SNAIL contained target sequences for miR‐370‐3p Figure  and was 3.5The above results indicated that circSWT1 affects tumor progression and EMT and that circSWT1 can increase SNAIL expression by sponging miR‐370‐3p. SNAIL has been linked to tumor development and EMT in previous researches.Furthermore, we conducted several experiments to evaluate the effects of circSWT1 in the development of NSCLC tumors. The findings revealed that circSWT1 had no influence on the viability, invasion, or migration of A549‐circSWT1‐SNAIL‐KO and H1299‐shcircSWT1‐SNAIL‐KO cells Figure . Overall3.6Using in vitro experiments, we looked at the involvement of circSWT1 in NSCLC tumor growth and EMT, and then we constructed mouse models to confirm the role of circSWT1 in vivo. First, subcutaneous injections of A549  and H1299  cells were given to nude mice. The results demonstrated that overexpression of circSWT1 in A549 cells promoted tumor growth. In contrast, knockdown of circSWT1 in H1299 cells inhibited tumor growth Figure . Further4CircRNAs were discovered nearly 50 years ago,Previous research has shown that circRNAs play a significant role in the progression of solid tumors, including NSCLC. Moreover, little is currently known about circSWT1. As a result, in four pairings of NSCLC tumor and normal tissues, we detected circRNAs that were derived from SWT1. Tumor tissues usually have high levels of circSWT1, while paired normal tissues have lower levels. Importantly, high levels of circSWT1 decreased overall survival rates and postoperative recurrence rates in NSCLC patients. In addition, we discovered that circSWT1 not only affects tumor, invasion, and metastasis but also induces EMT by acting on the miR‐370‐3p/SNAIL axis in NSCLC. Moreover, the nude mouse subcutaneous tumor model confirmed that circSWT1 can promote tumor growth. We concluded, based on the aforementioned findings, that circSWT1 could promote tumor progression and EMT via the miR‐370‐3p/SNAIL axis in NSCLC.Numerous researches had been conducted to investigate the biological activities of circRNAs.EMT is closely related to tumor metastasis, and Snail is a crucial EMT inducer. EMT is initially triggered in tumor cells, which then separate from the tumor and enter the circulation to promote metastasis. This process has been seen in several human malignancies, including breast,Snail is a critical target for pharmaceutical agents' development. Given the significant roles of Snail in many types of tumors, Snail inhibitors may be candidates to inhibit tumor progression and recurrence. According to earlier studies, Co (III)‐Ebox is a potent inhibitor of Snail‐mediated transcriptional suppression in breast cancer cells. Zinc finger domain‐containing Ebox‐binding proteins like Snail are inhibited by Co (III)‐Ebox, which reduces Snail activity without changing Snail protein levels. These findings revealed that this Co (III)‐DNA conjugate has beneficial therapeutic effects as an inhibitor of Snail‐induced tumor development and recurrence.In this study, we discovered that the level of circSWT1 was highly elevated in NSCLC and that a high level of circSWT1 was linked to a poor prognosis in NSCLC patients. Through the use of multivariate Cox analysis, circSWT1 was discovered to be a standalone predictor of the prognosis of NSCLC patients. According to these results, circSWT1 could serve as a prognostic biomarker for NSCLC. Additionally, circSWT1 overexpression facilitated NSCLC invasion, migration, and EMT both in vivo and in vitro. Moreover, knockdown of circSWT1 prevented the invasion, migration, and EMT processes of NSCLC in vivo and in vitro. Mechanistically, circSWT1 relieved the inhibition of downstream SNAIL by sponging miR‐370‐3p. Moreover, the biological function of circSWT1 relies on downstream SNAIL, which was verified by knocking out SNAIL in A549 and H1299 cells. SNAIL has been widely studied as a therapeutic target, and inhibitors of SNAIL such as Co (III)‐Ebox and CYD19 have been widely used. Thus, circSWT1 may also be a potential therapeutic target for NSCLC.5As a result of this work, circSWT1 was identified as a possible prognostic biomarker for NSCLC and was shown to have a crucial role in the progression and EMT of NSCLC. circSWT1 may also represent a fresh therapeutic target for NSCLC.Xiang Long: Conceptualization (supporting); data curation (lead); investigation ; methodology ; resources (supporting); supervision (supporting); validation (lead); writing – original draft . Ding‐Guo Wang: Data curation (supporting); formal analysis ; investigation ; software ; visualization (lead); writing – original draft . Zhi‐Bo Wu: Data curation (supporting); formal analysis (supporting); software ; validation (supporting); visualization (supporting). Zhong‐Min Liao: Investigation (supporting); software ; visualization (supporting). Jian‐Jun Xu: Conceptualization (lead); funding acquisition (lead); methodology ; project administration (lead); resources (lead); supervision (lead); writing – review and editing (lead).The following grants helped to fund this research: the National Natural Science Foundation of China (82060064).The authors declare that they have no competing interests.The Second Affiliated Hospital of Nanchang University Research Ethics Committee granted ethical permission (NO.SYXK2015‐0001), and each patient provided written informed consent.Not applicable.Figure S1.Click here for additional data file.Tables S1–S5.Click here for additional data file.Appendix S1.Click here for additional data file.


Evidence links gamma‐glutamyl transferase (GGT) to mortality in the general population. However, the relationship of GGT with all‐cause and cause‐specific mortality risk has been little explored in type 2 diabetes mellitus (T2DM) patients.We recruited 20 340 community‐dwelling T2DM patients between 2013 and 2014 in Jiangsu, China. Cox regression models were used to assess associations of GGT with all‐cause and specific‐cause mortality. Restricted cubic splines were used to analyze dose–response relationships between GGT and mortality. Stratified analysis was conducted to examine potential interaction effects by age, sex, smoking status, body mass index (BMI), diabetes duration, and dyslipidemia.p for interaction <.05), and HRs were stronger in the BMI <25 kg/m2 group and those without dyslipidemia.During a median follow‐up period of 7.04 years (interquartile range: 6.98–7.08), 2728 deaths occurred, including 902 (33.09%) due to cardiovascular disease (CVD), and 754 (27.58%) due to cancer. GGT concentrations were positively associated with all‐cause, CVD, and cancer mortality. Multivariable hazard ratios (HRs) for the highest (Q5) vs. the lowest quintile (Q1) were 1.63  for all‐cause mortality, 1.87 (95% CI: 1.49–2.35) for CVD mortality, and 1.43 (95% CI: 1.13–1.81) for cancer mortality. Effect modification by BMI and dyslipidemia was observed for all‐cause mortality  concentrations.2 and those with dyslipidemia.Stronger effects of GGT levels and all‐cause mortality were observed in the group with body mass index <25 kg/mGGT could potentially be used to predict the risk of cardiovascular disease and cancer among people with type 2 diabetes. According to the International Diabetes Federation, the global diabetes prevalence in 2021 was approximately 10.5% (536.6 million people), with the vast majority having type 2 diabetes mellitus (T2DM).Serum GGT is commonly used as a nonspecific marker of liver dysfunction and a biological marker of alcohol consumption.22.1Participants were enrolled from the Comprehensive Research on the Prevention and Control of the Diabetes (CRPCD) project in Jiangsu, China. In brief, the CRPCD project started in 2013, and 20 340 inhabitants were involved at baseline. A standardized questionnaire was used to collect information on demographic characteristics, self‐reported chronic disease history, and lifestyle factors. In addition, physical measurements and blood samples were taken at baseline. In the current analysis, we excluded 287 participants with important information lacking, 40 without GGT data, and 52 with chronic liver disease at recruitment. Therefore, a total of 19 961 participants were included in the final analysis.2.2Blood samples were collected in the morning after overnight fasting. All samples were analyzed in the laboratory at KingMed Diagnostics . Serum GGT was measured by the enzymatic rate method  Ltd).2.3International Classification of Diseases (ICD‐10).Participants were observed from the date of enrolment until 31 December 2020. Causes of death was obtained from the death certification system of the Jiangsu Provincial Center for Disease Control and Prevention. This system provides complete and accurate specific‐cause death data that have been medically validated. The primary outcomes of the current study included all‐cause mortality (A00‐Z99), CVD mortality (I00‐I79), and cancer mortality (C00‐C97). In addition, deaths due to CVD were divided into the following subgroups: CHD (I20‐I25) and stroke (I60‐I64). Cancer deaths were divided into the following subgroups: esophagus cancer (C15), gastric cancer (C16), intestinal cancer (C18‐C20), liver cancer (C22), pancreatic cancer (C25), and lung cancer (C34), which were coded using the 10th revision of the 2.4A smoker was defined as having smoked more than 100 cigarettes in his or her lifetime.2.5n, %). The distribution of GGT values was right skewed, therefore a natural‐log transformed GGT was applied. GGT values were categorized into five groups using the 20th (reference), 40th, 60th, and 80th percentiles as cut‐points; the cutoff points corresponded to 17, 23, 31, and 48 U/L. Person‐time was calculated for each individual from the date of recruitment to the date of death or the date of last follow‐up (31 December 2020).Baseline characteristics were described by means (SD) for continuous variables and proportions for categorical variables  and 95% confidence intervals (CIs) were calculated according to quintiles of log‐transformed GGT concentrations. Model 1 was adjusted for age at blood draw (continuous) and sex. Model 2 was further adjusted for education, income, smoking status, alcohol consumption, BMI (continuous), physical activity (continuous), and lipid‐lowering medication use. All‐cause and CVD mortality were additionally adjusted for self‐reported stroke and CHD at baseline. Model 3 was further adjusted for diabetes duration (continuous), antidiabetic medication use and insulin use.Continuous variables were compared between groups with independent n = 20) was truncated to avoid the effect of outliers on the curve. p for nonlinearity <.05 was considered nonlinearity, and p for linearity <.05 was considered linearity.To assess linearity in the associations of serum GGT concentration with the risk of all‐cause and specific‐cause mortality in the fully adjusted model, we performed restricted cubic spline analysis with four knots and calculated the likelihood ratio test by comparing the model with only the linear term of these markers to the model with both the linear and cubic spline terms. The top 0.1% of GGT concentrations (2), smoking status , median diabetes duration , and dyslipidemia . Dyslipidemia was defined by lipid indicators  or hospital diagnosis. To investigate potential effect modification by these stratification variables, we used a likelihood ratio test comparing the models with and without interaction terms between GGT concentrations and each of the stratification variables. Considering that weight change is a typical symptom in T2DM progression, we further analyze the associations of serum GGT concentrations with all‐cause and cause‐specific mortality among nonobese (BMI <30 kg/m2) T2DM patients.We also conducted stratified analysis according to age , sex , BMI , participants who died due to accident (ICD‐10: V00‐Y98) (n = 153), or participants who used lipid‐lowering medicine (n = 2081). Additionally, considering the disruption caused by the COVID‐19 pandemic, we changed the date of the last follow‐up to 31 December 2019 (before the start of the COVID‐19 pandemic) for sensitivity analysis.Sensitivity analysis was performed by excluding participants in the first 2 years of follow‐up . All statistical tests were two sided, and 3The median follow‐up period was 7.04 years (interquartile range: 6.98–7.08 years). Among 19 961 participants, 2728 deaths occurred, including 902 (33.09%) deaths due to CVD and 754 (27.58%) deaths due to cancer. The distribution of GGT is shown in Figure S3.1p for nonlinear = .51 and p for linear <.0001).As shown in Table 3.2p for nonlinear = .04).Higher GGT concentrations were associated with an increased risk of CVD mortality . The association remained stable after further adjustment for diabetes duration, antidiabetic medicine, and insulin use (Table 3.3p for nonlinear <.0001)  Table . For can2 group and those without dyslipidemia. (both p for interaction <.05). Furthermore, among nonobese T2DM patients, serum GGT concentration was also positively associated with the risk of all‐cause, CVD, and cancer mortality , and they were thus well represented. In addition, serum GGT detection was completed in the same laboratory with unified methods and instruments, effective controls of measurement bias. However, several limitations need to be considered. First, although patients with known hepatic disease were excluded, the prevalence of NAFLD was unknown due to a lack of ultrasound liver examination, which did not allow us to evaluate the role of NAFLD in the associations. Second, there may be regression dilution bias using the baseline GGT concentration, so the one‐time collection of baseline information may not enable us to assess changes throughout the whole follow‐up period. Third, the present study lacked data on the time of smoking initiation and how much was smoked as well as changes in income levels during COVID‐19, which may have an effect on mortality. Finally, the mortality surveillance system may underreport and misclassify, though underreporting surveys are performed every year for the database, with automatic evaluation of misclassifications. Misclassification and underreporting were <5%.5In conclusion, our findings suggest that, in the Chinese T2DM population, elevated serum GGT concentration is associated with higher mortality for all‐cause, CVD, and cancer. Future studies should focus on revealing the role of obesity, NAFLD, and lipids in this association.Conception and design of the study: Ming Wu, Jian Su, Haoyu Guan, Jinyi Zhou, Chong Shen. Collection of data: Hao Yu, Yu Qin, Zheng Zhu, Jie Yang, Enchun Pan, Yan Lu, Jinyi Zhou. Data analysis, interpretation of results, and writing of the manuscript: Haoyu Guan, Ke Liu, Xikang Fan, Jian Su. All the authors revised the manuscript critically for its intellectual content and approved the final version.This work was supported by grants from Jiangsu Province Leading Talents and Innovation Team Program (No. K201105); Jiangsu Provincial Fifth “333 Project” (BRA2020090); Jiangsu Provincial Health Commission 2020 Medical Research Project Approval (M2020085); the Suzhou Program of Science & Technology Development (SS202010).The authors declare that they have no conflicts of interest.Ethics approval for this study was granted by the Ethics Board of Jiangsu Provincial Center for Disease Control and Prevention (No. 2013026). All participants provided written informed consent, and the methods were carried out in accordance with relevant guidelines and regulations.Data S1. Supplementary FileClick here for additional data file.


Human placental mesenchymal stem cells (PMSCs) can prevent liver ischaemia–reperfusion injury (LIRI). However, their therapeutic effects are limited. Therefore, additional research is required to elucidate the mechanisms of PMSC‐mediated LIRI prevention and enhance the related therapeutic effects. This study aimed to examine the role of the Lin28 protein in the regulation of glucose metabolism in PMSCs. Further, it explored whether Lin28 could enhance the protective effects of PMSCs against LIRI and investigated the underlying mechanisms. Western blotting was performed to examine Lin28 expression in PMSCs under hypoxic conditions. A Lin28 overexpression construct was introduced into PMSCs, and the effect on glucose metabolism was examined using a glucose metabolism kit. Further, the expression of some proteins involved in glucose metabolism and the PI3K‐AKT pathway and the levels of microRNA Let‐7a–g were examined using western blots and real‐time quantitative PCR, respectively. To examine the relationship between Lin28 and the PI3K‐Akt pathway, the effects of AKT inhibitor treatment on the changes induced by Lin28 overexpression were examined. Subsequently, AML12 cells were co‐cultured with PMSCs to elucidate the mechanisms via which PMSCs prevent hypoxic injury in liver cells in vitro. Finally, C57BL/6J mice were used to establish a partial warm ischaemia–reperfusion model. The mice received intravenous injections containing PMSCs (control and Lin28‐overexpressing PMSCs). Finally, their serum transaminase levels and degree of liver injury were assessed using biochemical and histopathological methods, respectively. Lin28 was upregulated under hypoxic conditions in PMSCs. Lin28 exerted protective effects against hypoxia‐induced cell proliferation. Moreover, it increased the glycolytic capacity of PMSCs, allowing PMSCs to produce more energy under hypoxic conditions. Lin28 also activated the PI3K‐Akt signalling pathway under hypoxic conditions, and its effects were attenuated by AKT inhibition. Lin28 overexpression could protect cells against LIRI‐induced liver damage, inflammation and apoptosis and could also attenuate hypoxia‐induced hepatocyte injury. Lin28 enhances glucose metabolism under hypoxic conditions in PMSCs, thereby exerting protective effects against LIRI by activating the PI3K‐Akt signalling pathway. Our study is the first to report the potential of genetically modified PMSCs for LIRI treatment. Owing to this switch, called the Warburg effect, cancer cells show unique mitochondrial utilization.In the present study, a partial warm ischaemia–reperfusion injury mouse model and hypoxia‐treated hepatocyte model were used to examine whether Lin28 can increase the glycolytic capacity of PMSCs under hypoxic conditions and protect liver cells against LIRI. Further, the involvement of PI3‐Akt signalling in the effects of Lin28 on PMSC glycolysis was also explored.22.1We purchased male C57BL/6J mice (6–8 weeks old) from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed in the Animal Facility of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. All animal experiments were conducted after approval from the Institutional Animal Care and Use Committee of the Tongji Medical College, Huazhong University of Science and Technology. All procedures complied with the relevant guidelines and regulations of the Chinese Council on Animal Care.2.27 cells/mL) 1 h prior to the induction of hepatic ischaemia. After 1 h of ischaemia and 6 h of reperfusion, mice were sacrificed, and serum and liver tissue samples were collected.Male C57BL/6J mice (20–22 g) were randomly assigned to one of four groups: Sham; LIRI; LIRI + PMSC; and LIRI + PMSC‐Lin28. A partial LIRI model was developed using methods described in an earlier study.2.3Blood collected from mice was first chilled on ice (30 min) and then centrifuged at 8000 RPM (15 min). The supernatant was diluted as appropriate and a standard automatic analyser (Mindray BS‐200) was used to detect serum alanine transaminase (ALT) and aspartate transaminase (AST) levels.2.42 was used to maintain a normoxic environment. In contrast, a hypoxia incubator (Whitley H35 Hypoxystation) containing 5% CO2, 1% O2 and 94% N2 was used for maintaining an anoxic environment. To establish the ischemic and hypoxic cellular models, cells were cultured to a density of approximately 70%. Subsequently, the old medium was removed, and serum‐free medium was added; cells were incubated in anoxic conditions for 24 h. Additionally, cells in the inhibitor treatment group were treated with MK2206 . Four treatment groups were established: PMSC, PMSC‐Lin28, PMSC + MK2206 2Hcl and PMSC‐Lin28 + MK2206. We maintained AML12 cells under hypoxic conditions for 24 h to construct a cellular model of ischaemia and hypoxia. The cells were divided into three groups: the AML12 group , AML12 + PMSC group (AML12 cells co‐cultured with PMSCs) and AML12 + PMSC‐Lin28 group (AML12 cells co‐cultured with PMSCs‐Lin28).Human PMSCs (Shanghai Tongji University) and murine hepatocyte cells  were cultured in Dulbecco's modified eagle medium/F‐12 (Hyclone) supplemented with 10% foetal bovine serum (Hyclone), 40 ng/mL dexamethasone , and ITS Liquid Media Supplement . The AML12 cells and PMSCs were placed in normoxic and anoxic environments, respectively, at 37°C. A humidified incubator containing 5% CO2.5When cells reached a density of approximately 70%, they were cultured in OPTI‐MEM Reduced Serum Medium (Gibco) with a Lin28 overexpression lentivirus  for 72 h. Subsequently, fluorescence microscopy was used to detect transfection efficiency.2.6Glucose , lactic acid , ATP  and NADPH  detection kits were used to detect intracellular glucose levels, LA levels, ATP levels, and the NADPH/NADP+ ratio, respectively. ECAR was detected using the Seahorse XF Glycolysis Stress Test Kit .2.72.7.1After 6 h of ischaemia, liver tissue was fixed in 4% paraformaldehyde. The tissue was dehydrated using an increasing ethanol concentration gradient and embedded in paraffin. Subsequently, 4‐μm‐thick sections were obtained and stained with Haematoxylin and Eosin. The stained samples were examined by two independent pathologists, who assessed hepatic necrosis based on Suzuki's scores.2.7.2A TUNEL Kit  was used based on manufacturer's instructions. First, tissue sections were deparaffinized using protease K. Subsequently, membranes were disrupted, the reaction solution was added and nuclei were stained with DAPI. Finally, the sections were imaged using an upright fluorescence microscope (NIKON ECLIPSE C1). The proportion of TUNEL‐positive cells was calculated using five randomly selected fields.2.7.3Myeloperoxidase (MPO) immunohistochemistry was performed using mouse anti‐MPO  and HRP‐labelled goat anti‐mpO  antibodies and the histochemical DAB chromogenic agent kit . All procedures were performed based on manufacturer's instructions. The fixed tissue was first blocked with serum. Subsequently, the primary antibody, secondary antibody and developer were added. Finally, the nuclei were stained with DAPI. Then, the tissue was examined under a microscope, and images were captured and analysed. The haematoxylin‐stained nuclei appeared blue, whereas positive mPO expression was indicated by a brownish yellow colour.2.8The CCK‐8 kit (Dojindo) was used to detect cell viability. Before hypoxia treatment, 10,000 cells were planted in each well of a 96‐well plate, and three holes were made in each well. After 24 h of culture, the medium was replaced and 10 μL of the CCK‐8 reagent was added to each well . Finally, a microplate reader was used to measure the absorbance at 450 nm.2.9An Annexin V‐FITC and propidium iodide staining kit (MULTISCIENCES) was used to examine cell apoptosis using manufacturer's instructions. Using a Flow Cytometer (BD FACSCelesta) and FlowJo software for data analysis, we measured the percentages of apoptotic cells.2.10GADPH and U6 were chosen as internal controls for mRNA and microRNA, respectively. Each experiment was performed at least in triplicate. Primer sequences are shown in the table below  was used to isolate the total mRNA from PMSCs. The mRNA was then reverse transcribed using a high‐capacity cDNA reverse transcription kit (TAKARA) based on manufacturer's instructions. Target gene expression was evaluated with qRT‐PCR using a SYBR Green kit (TAKARA). StepOne software (Thermo Fisher Scientific) was used to analyse the data. For examining microRNA levels, the high‐capacity microRNA reverse transcription kit (TAKARA) was used; the other steps remained the same as those for mRNA analysis. ow Table . Relativ2.11IP lysis buffer (Beyotime) with protease inhibitors was used to extract the total protein from cell lysates or tissue sample. A microplate reader (BioTek) and BCA Protein Assay Kit (Beyotime) were used to measure the protein concentration. Proteins (80 μg) were separated using 10% SDS‐PAGE and transferred onto polyvinylidene fluoride membranes . The membranes were blocked with 5% skim milk in TBST and then incubated with primary antibodies for 16 h. Antibodies against β‐catenin , lactate dehydrogenase A , AKT , pAKT (ser473), Lin28 , total OXPHOS , Hexokinase I , Hexokinase II , M2‐pyruvate kinase , 3‐phosphoinositide dependent kinase‐1  and 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase 2  were used. Subsequently, the membranes were blocked with horseradish peroxidase‐conjugated goat anti‐rabbit IgG H&L or goat anti‐mouse IgG H&L antibody  at room temperature (20–25°C) for 1 h. The ECL reagent (Beyotime) was used to detect protein bands. The Chemiluminescence Imaging System (GeneGnome) and Image‐Pro Plus software (Media Cybernetics) were used for protein quantification. Proteins levels were normalized based on β‐catenin levels, and all experiments were performed at least thrice.2.12anova was used to compare multiples groups, whereas unpaired t‐tests were used to compare two groups. p‐values < 0.05 were considered statistically significant.All statistical analyses were performed using Prism software . All data were expressed as the mean ± standard error of the mean. One‐way 33.1A PMSC hypoxia model was used to examine the expression of Lin28 in PMSCs under hypoxic conditions. We found that hypoxia obviously increased Lin28 expression in PMSCs Figure . The Lin3.2In order to understand the pathways through which Lin28 increases the glycolytic capacity of PMSCs, we examined the levels of glycolysis‐related proteins in these cells under normoxic and hypoxic conditions. The proteins LDHA, PDK1, PKM2, HEX I, HEX II and PFKFB2 – which are enzymes involved in the regulation of glycolysis – were upregulated during hypoxia. However, these proteins, except LDHA, showed no significant difference in expression in PMSCs overexpressing Lin28 Figure . The mit3.3PI3K and LDHA were quantified in PMSCs and PMSCs‐Lin28 under normoxic and hypoxic conditions using qRT‐PCR. Moreover, the protein expression of AKT, pAKT (ser473), Lin28, and LDHA was detected using western blots. The transcriptional levels of PI3K and LDHA were higher in the PMSCs‐Lin28 group than in the PMSCs group. These levels were also higher under hypoxic conditions than under normoxic conditions  has a region that can bind to microRNAs from the Let7 family. We speculated that members of the microRNA Let7 family bind to the promoter region of PIK3CA and inhibit its transcription. This decreases the expression of PI3K, consistent with our previous results. We aim to explore these mechanisms in subsequent studies.Although Lin28 is known to activate the PI3K‐Akt pathway,5In conclusion, our study shows that Lin28 can improve the glycolytic capacity of PMSCs under hypoxic conditions via the PI3K‐Akt pathway. Therefore, it can promote energy production, even under hypoxia, thus increasing the protection PMSCs offer against LIRI. To our knowledge, our study is the first to show that Lin28 alters glucose metabolism in PMSCs via the PI3K‐Akt pathway, improving LIRI prevention. Despite its limitations, our study provides new insights into the molecular regulation of glycolysis in PMSCs. These insights pave the way for more effective MSC therapies and strategies for LIRI prevention.Xi Zhou: Data curation (lead); formal analysis (lead); methodology (lead); writing – original draft (lead). Junbo Li: Data curation ; formal analysis ; methodology ; visualization . Jin Wang: Conceptualization ; data curation ; investigation ; visualization . Huifang Yang: Conceptualization ; software . xiaoyun xie: Conceptualization ; writing – review and editing . Zhishui Chen: Formal analysis ; funding acquisition ; writing – review and editing . Bo Zhang: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; writing – review and editing .This work was funded by the National Natural Science Foundation of China (81800580 and 81770652).The authors have declared that no conflict of interest exists.


This study aimed to investigate the association between a novel kidney disease index (KDI), which combines information from both estimated glomerular filtration rate (eGFR) and urinary albumin‐to‐creatinine ratio (uACR), and all‐cause and cardiovascular disease (CVD) mortality among individuals with hypertension.We analyzed data from 19 988 adults with hypertension who participated in the National Health and Nutrition Examination Survey from 1999 to 2018. Mortality outcomes were determined by linking to National Death Index records through December 31, 2019. Cox proportional hazards models were used to estimate hazard ratios and 95% confidence intervals for all‐cause and CVD mortality.p value for trend <.05). During a follow‐up period of 179 859 person‐years, a total of 5069 deaths were documented, including 1741 from cardiovascular causes. After multivariable adjustment, each standard deviation increment in KDI level was associated with a 27% increased risk of all‐cause mortality and a 31% increased risk of cardiovascular deaths (both p < .05). Further analysis showed a J‐shaped association between KDI and mortality, with the risk increasing dramatically when KDI exceeded 0.27.Baseline KDI levels were positively associated with glucose, insulin resistance, hemoglobin A1c, triglycerides, and C‐reactive protein (Elevated KDI levels were significantly associated with increased mortality from all causes and CVD among individuals with hypertension. We recommend routine testing of eGFR and uACR in hypertensive patients, and using KDI as a tool to identify individuals who are most likely to benefit from preventive therapies. ASCVDatherosclerotic cardiovascular diseaseCKDchronic kidney diseaseCRPC‐reactive proteinCVDcardiovascular diseaseeGFRestimated glomerular filtration rateHDLhigh‐density lipoproteinHOMA‐IRhomeostatic model assessment of insulin resistanceHTNhypertensionKDIkidney disease indexLDLlow‐density lipoproteinNHANESNational Health and Nutrition Examination SurveyRAASrenin–angiotensin–aldosterone systemTCtotal cholesterolTGtotal triglycerideuACRurine albumin‐to‐creatinine ratio1Hypertension (HTN) is a highly prevalent disorder that affects a vast majority of adults worldwide during their lifetimes. The global prevalence of HTN is estimated to be 26%, and this is expected to increase to 29% by 2025, driven largely by increases in economically developing nations.The urine albumin‐to‐creatinine ratio (uACR) and the estimated glomerular filtration rate (eGFR) are well‐known independent risk factors for CVD and mortality among individuals with HTN.Therefore, we conducted a prospective study to investigate the associations between KDI and all‐cause and CVD mortality in a nationally representative sample of adults in the United States with HTN.22.1https://www.cdc.gov/nchs/nhanes). In all, 101 316 participants were included in 10 consecutive circles from 1999 to 2018. We first excluded individuals without follow‐up records (n = 42 252), and then participants without diagnosed HTN were excluded (n = 35 909). In addition, we excluded individuals aged less than 18 years old (n = 118) or pregnant individuals (n = 153). This is an illustration of our exclusion criteria. Details can be found in the flow diagram . A total of 19 988 were finally included in the current study. HTN was defined as meeting one of the following criteria: self‐reported doctor diagnosis of HTN, use of anti‐HTN medication, or systolic blood pressure of 140 mmHg or higher, or diastolic blood pressure of 90 mmHg or high, with more than three measurements. The entire process of data selection is presented in Figure The current prospective study was based on the National Health and Nutrition Examination Survey (NHANES), which is a nationwide representative survey designed to collect information on health and nutrition in the United States. Data were collected through structured interviews at home and mobile center. The entire study was approved by the Centers for Disease Control and Prevention's Institutional Review Board. Informed permission was obtained from all participants. Detailed information on the survey's design and methodology can be found on the NHANES website  Serum Creatinine 2009 equation due to its better accuracy compared to the Modification of Diet in Renal Disease equation. KDI was the geometric mean of 1/eGFR and natural log‐transformed (uACR × 100). Furthermore, KDI was categorized into quartiles: Q1 (≤0.26), Q2 (0.26–0.29), Q3 (0.29–0.33), and Q4 (>0.33).2.3All‐cause mortality was defined as death due to any cause during follow‐up according to the records of the National Death Index before December 31, 2019. CVD mortality was defined as International Classification of Diseases, Tenth Revision codes I00–I09, I11, I13, I20‐I51, or I60–I69.2.4We included various covariates that may affect the outcome, including age, sex, ethnicity, education level, family poverty income ratio, smoking and drinking status, comorbid diseases, and antihypertensive drug use. The definition of hyperlipidemia was based on meeting at least one of the following criteria: (1) self‐reported doctor‐diagnosed hyperlipidemia; (2) total triglyceride (TG) levels ≥150 mg/dL, total cholesterol (TC) levels ≥200 mg/dL, high‐density lipoprotein (HDL) levels <40 mg/dL, or low‐density lipoprotein (LDL) levels ≥130 mg/dL; or (3) the use of antihyperlipidemic medication. Atherosclerotic cardiovascular disease (ASCVD) was defined as coronary heart disease, heart attack, angina, or stroke. Creatinine, uACR, plasma glucose, insulin, hemoglobin A1C (HbA1c), TG, TC, HDL, LDL, and C‐reactive protein (CRP) were measured at recruitment when the participants provided their blood samples. The NHANES website provided detailed procedures in collecting blood sample measurements.2.5H‐test, or χ2 test.In light of NHANES' utilization of a complex, multistage, probability sampling methodology to identify representative participants,We utilized multivariate Cox proportional hazards regression to analyze the relationship between KDI and all‐cause and CVD mortality, to estimate the hazard ratios (HRs) and corresponding 95% confidence intervals (CIs). The reference group for our analysis was Q1, and we assigned a median value to each category to determine if there was a linear trend.We employed restricted cubic spline regression with four knots  and multivariable adjustment to evaluate the correlation between KDI and all‐cause and CVD mortality. We conducted a likelihood ratio test to determine nonlinearity. If nonlinearity was detected, we constructed two piecewise Cox proportional hazards regression models using the inflection point.p value less than .05. All statistical analyses were performed using R 4.2 .We further conducted stratified analyses based on age, gender, ethnicity, drinking status, BMI, HTN medication use, diabetes, hyperlipidemia, and ASCVD. Additionally, we assessed potential interactions between KDI and the various stratification factors. To ensure the robustness of our findings, we conducted a sensitivity analysis. First, we excluded participants who died within 2 years of follow‐up to reduce the potential for reverse causation bias. Second, we performed further adjustments for blood lipids, HOMA‐IR, HbA1c, and CRP. Statistical significance was defined as a two‐sided 32, and uACR was 63.82 (3.20) mg/g. The baseline characteristics of the study population according to KDI are presented in Table The study population consisted of 19 988 adults diagnosed with HTN, with a mean (SE) age of 56.82 (0.21) years. Of these, 9924 were males  and 10 064 were females . The weighted mean (SE) eGFR was 84.67 (0.28) mL/min/1.73 mp values for trend <.05). Moreover, higher levels of KDI were associated with lower baseline levels of HDL.The least‐square means of cardiometabolic biomarkers according to KDI are presented in Table p value for trend <.05). Additionally, the corresponding HRs and 95% CIs for cardiovascular death were 1.00 (reference), 0.80 , 1.19 , and 2.16 , respectively . Furthermore, for each standard deviation increase in KDI level, there was a 27% increased risk of all‐cause mortality and a 31% increased risk of cardiovascular deaths , with the inflection point at 0.27.Over a follow‐up period of 179 859 person‐years, a total of 5069 deaths were recorded, including 1741 from cardiovascular causes. After adjusting for age, gender, ethnicity, BMI, lifestyle factors, and the presence of chronic disease, our analysis revealed that higher KDI levels were significantly associated with increased all‐cause and cardiovascular mortality. As presented in Table 16 1.46, .20, respp value for interaction ≥.05).The results were consistent when we conducted stratified analyses by age, sex, ethnicity, BMI, drinking status, prediabetes or diabetes, hyperlipidemia, and ASCVD (Supporting Information: Table In our sensitivity analysis, we excluded participants who died within 2 years of follow‐up (Supporting Information: Table 4We conducted a large prospective cohort study on US adults with HTN and found that an elevated KDI was significantly associated with higher mortality outcomes, including all‐cause mortality and cardiovascular deaths, independent of established risk factors such as age, gender, ethnicity, BMI, smoking or drinking status, prediabetes or diabetes, hyperlipidemia, ASCVD, and antihypertensive drug use. Stratifying our analysis by age, gender, ethnicity, BMI, antihypertensive drug use, and comorbidity showed consistent findings in each stratum. Further subgroup analysis adjusting for blood lipids, HOMA‐IR, HbA1c, and CRP did not significantly change the results. Sensitivity analysis, excluding participants who died within 2 years, yielded similar findings. Elevated KDI was also associated with higher levels of cardiometabolic biomarkers, such as glucose, HOMA‐IR, HbA1c, TG, and CRP. Restricted cubic spline analysis showed a J‐shaped association between KDI and mortality among individuals with HTN, with an inflection point of 0.27. When KDI exceeded 0.27, the risk of all‐cause and CVD mortality significantly increased. Our findings suggest that incorporating KDI into cardiovascular risk prediction can be a useful tool for identifying hypertensive patients at higher risk of mortality and initiating appropriate interventions.HTN serves as a prevalent etiological factor for renal impairment, resulting in a decline in eGFR and an elevation in uACR. Nonetheless, prior investigations have predominantly approached eGFR and uACR as distinct entities, seldom combining them into a composite variable for comprehensive analysis. Indeed, previous studies have shown that eGFR and uACR are independent risk factors of all‐cause mortality in hypertensive patients.Using geometric mean to combine continuous variables into a composite variable, as suggested by Gerstein et al.,Although the exact mechanisms underlying the association between eGFR and albuminuria and CVD are not fully understood, evidence from animal and human studies suggests that albuminuria is associated with abnormalities in endothelial glycocalyx and other endothelial structures, which may lead to inflammation and endothelial dysfunction.Prior investigations have generated divergent findings concerning the capacity of eGFR and uACR to enhance CVD prediction beyond conventional models. Chang et al.Further research is needed to determine the optimal management strategies for patients with KDI levels exceeding 0.27, such as more intensive blood pressure targets, earlier initiation of RAAS blockade, or more frequent monitoring of kidney function. Additionally, future studies should evaluate whether interventions targeting KDI levels can improve cardiovascular outcomes in hypertensive patients.Our study has several strengths, including large sample size, a prospective design, and comprehensive data collection. However, there are several limitations that need to be acknowledged. Firstly, the observational study design does not allow us to establish causality. Secondly, using a single measurement for KDI may not accurately reflect long‐term levels and changes over time. Thirdly, changes in covariates over time may have attenuated the true association between KDI levels and mortality. Fourthly, we did not have detailed information on the severity of HTN. Finally, the possibility of residual or unknown confounding cannot be entirely ruled out.5Our findings demonstrate a significant positive association between KDI, a composite variable derived from both eGFR and uACR, and increased risk of all‐cause and CVD mortality in hypertensive patients. Notably, we observed a J‐shaped relationship between KDI and mortality outcomes, with an incremental increase in mortality risk when KDI exceeded 0.27. These results highlight the potential clinical significance of KDI in predicting adverse outcomes, including mortality, in hypertensive patients. We recommend that eGFR and uACR be routinely tested in hypertensive patients, in addition to current HTN guidelines, and that KDI be used as a tool to identify individuals at the highest risk who are most likely to benefit from preventive therapies.The authors declare no conflict of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


For 4 weeks, the KMP group received a capsule containing 50‐mg KMP daily, a dose approximately five times higher than the estimated human dietary intake. The placebo group received a capsule containing cornstarch‐based powder daily. The general toxicity parameters were evaluated by examining the characteristics of the participants, hematological and blood biochemical parameters, general urinalysis, qualitative urine tests, and adverse events. No clinical changes were observed in anthropometric and blood pressure measurements or blood and urine parameters in the KMP group compared to those in the placebo group. Furthermore, no adverse events owing to KMP aglycone administration occurred. The study results revealed that the consumption of 50‐mg KMP aglycone daily for 4 weeks is safe in healthy adults.Kaempferol (KMP) is an important flavonoid in many fruits and vegetables. Preclinical studies on KMP have reported its pharmacological effects, including antimicrobial, antioxidant, anti‐inflammatory, antitumor, antidiabetic, myocardial protective, and neuroprotective effects. Additionally, some epidemiological studies have revealed a negative association between the consumption of KMP‐containing foods and the risk of developing several disorders, such as cancer and cardiovascular diseases. Thus, although a large body of literature has demonstrated the benefits of KMP supplementation, there are no reports of clinical trials evaluating the safety of KMP aglycone administration or KMP aglycone‐rich food consumption. The purpose of this study was to evaluate the safety of a high dose of KMP aglycone by administrating KMP aglycone‐containing supplements to healthy adults. This study had a randomized, double‐blind, placebo‐controlled design and a 4‐week duration. Participants were randomly allocated to the KMP ( A kaempferol aglycone‐based supplement was developed using horseradish leaves. This is the first clinical evaluation of the safety of kaempferol aglycone intake. A 4‐week continuous intake of 50‐mg kaempferol aglycone/day was safe. Among these compounds, flavonoids have garnered increasing attention owing to their antioxidant, cardioprotective, and immunomodulatory properties , a flavonol widely found in nature, was shown to enhance hypoxia‐induced HIF‐1α degradation and increase mitochondrial complex IV activity, thereby stimulating aerobic respiration and increasing ATP content in vitro and in vivo  leaves, containing KMP aglycone was obtained from Otsuka Pharmaceutical Co., Ltd.  and comprised approximately 16.1% KMP aglycone as an active ingredient , blood pressure (systolic/diastolic), and pulse rate. The height was measured only during the preliminary examination period, and the BMI was calculated based on this measurement. During the preliminary examination period, participants ingested a single dose of 25‐mg KMP, and urine was collected up to 24 h after supplementation to assess KMP excretion levels.2.4Urine samples were collected for up to 24 h after a single dose of 25‐mg KMP supplementation and stored at 4°C. All samples collected for each participant were pooled at the end of the 24‐h period. The volume of each sample was recorded, and samples were stored at −80°C until further analysis.2.5The urine samples (100 μL) were mixed with 100 μL of β‐glucuronidase solution, diluted in 0.2 M sodium acetate buffer (pH 5.0) for a final concentration of 50 units of β‐glucuronidase. After incubating the mixtures at 37°C for 0.5 h, 200 μL of 4% phosphate buffer containing apigenin‐d5  was added. Then, the urine samples were transferred to a conditioned 96‐well MCX‐Elute plate (Waters Corporation). After the samples were drawn through the sorbent bed, they were washed with 200 μL of 2% formic acid in water, followed by 200 μL of 40% methanol. KMP and the internal standard were eluted with 150 μL of methanol/acetonitrile (1:1) in another 96‐well elution plate for the final analysis.2.6Chromatographic separation of the injected 20‐μL sample was conducted using a reverse‐phased C18 analytical column , and liquid chromatographic separation was achieved using a high‐performance liquid chromatography (HPLC) system (Shiseido Nanospace). The mobile phase used was a mixture of 0.1% formic acid with water and acetonitrile, operated at a flow rate of 0.35 mL/min. The initial mobile phase composition (20% acetonitrile) was constant for 0.5 min, followed by a linear gradient to 95% acetonitrile for 1.5 min, maintained until 3.5 min, then changed to 20% acetonitrile at 3.6 min, and maintained until 5.5 min. Next, the samples separated using HPLC were analyzed using a Sciex API‐3000 tandem mass spectrometer equipped with an electrospray interface (Sciex).2.7The gender, age, BMI, and urinary KMP excretion rate measured at the preliminary examination were used as stratification factors. The patients were randomly assigned to the test or placebo group using the stratified block randomization method. There were no significant differences between the two groups regarding the measured parameters. The test group received KMP supplement capsules (50‐mg KMP aglycon/two capsules/day), whereas the placebo group received placebo capsules (cornstarch‐based powder/two capsules/day). The investigator ensured that the two capsule types were indistinguishable. The relevant identification codes were printed on the pack containing each substance, then printed and sealed in an envelope, and delivered to the study outsource provider. The identification codes were sealed in an envelope and sent to the allocator, who confirmed that neither of the capsules could be identified by their appearance and/or smell. Next, the allocator replaced the KMP supplement and placebo codes with another control code that could not be easily identifiable, thereby blinding the study. The products were transferred to the test product manager. The control codes were tightly sealed in an envelope together with the correspondence table and kept by the allocator until the study was unblinded.2.81c). For general urinalysis, pH and specific gravity were measured. For qualitative urine tests, protein, glucose, urobilinogen, bilirubin, ketone body, and occult blood presence were measured. These assessments were performed at the beginning of the study, 2 and 4 weeks after the intake initiation, and during the follow‐up observational period.Hematology tests included white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count (PLT). Biochemical examination of blood comprised the measurement of the values of total protein, albumin, total bilirubin, direct bilirubin, alkaline phosphatase (ALP), γ‐glutamyltranspeptidase (γ‐GTP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), creatine kinase (CK), C‐reactive protein (CRP), total cholesterol, high‐density lipoprotein cholesterol (HDL‐cho), low‐density lipoprotein cholesterol (LDL‐cho), triglyceride (TG), blood urea nitrogen (BUN), creatinine, uric acid (UA), sodium, potassium, chloride, calcium, phosphorus, glucose, and glycated hemoglobin , and statistical significance was set at 33.1Forty‐eight participants were enrolled in this trial, and all participants completed the trial Figure . The bas3.2Table 3.3No significant differences were observed between groups for WBC and RBC values throughout the study period Table . In the 3.41c values at follow‐up, in Cl values at weeks 2 and 4, and in K values at week 2 and follow‐up. In the KMP group, compared with baseline values, significant changes were observed in total protein, HDL‐cho, LDL‐cho, and UA values at week 4, in CK, Na, K, Ca, and glucose values at follow‐up, and in HbA1c values at week 4 and follow‐up. Total bilirubin, ALP, and K values at follow‐up and direct bilirubin values at week 2 and follow‐up were significantly higher in the KMP than in the placebo group. Conversely, Na at week 2, AST and ALT at week 4, P at weeks 2 and 4, and γ‐GTP values at week 4 and follow‐up were significantly lower in the KMP group than in the placebo group. The physician confirmed that all changes were not clinically relevant.The results of blood biochemistry tests are shown in Table 3.5Urinary pH values significantly increased in the placebo group at follow‐up than at baseline. Urinary pH was significantly higher in the KMP group than in the placebo group at week 2 Table . The qua3.6n = 4, 6 events; abdominal pain, dry eyes, stomatitis, headache, and cough); KMP group  (Table No side effects were attributable to the test powder in any participant. Adverse events occurred in both groups: placebo group (g) Table .4Although growing evidence supports the potential use of KMP for the prevention and/or treatment of several diseases, to our knowledge, no clinical trials have been conducted to verify these effects using KMP aglycone or KMP aglycone‐rich foods. This may be because KMP exists in nature mainly as glycosides and conjugates, and no food or food material contains a large amount of aglycone. Consequently, there are no clinical data on the safety of KMP intake. In vitro studies have reported the possibility of pro‐oxidant effects  with high KMP doses  ; data curation ; formal analysis ; investigation ; methodology ; visualization ; writing – original draft (lead). Tsubasa Mizokami: Data curation ; formal analysis ; investigation ; visualization . Hikaru Ito: Formal analysis ; investigation . Yasutaka Ikeda: Conceptualization ; data curation ; methodology ; writing – review and editing (lead).This research received no specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.All authors are employees of Otsuka Pharmaceutical Co., Ltd.All study procedures were approved by the ethics committee of AMC Nishi‐Umeda Clinic and were conducted in accordance with the standards of the 1964 Declaration of Helsinki and its later amendments.Informed consent was obtained from all participants included in the study.


The growing population of long‐term childhood cancer survivors encounter a substantial burden of cardiovascular complications. The highest risk of cardiovascular complications is associated with exposure to anthracyclines and chest radiation. Longitudinal cardiovascular surveillance is recommended for childhood cancer patients; however, the optimal methods and timing are yet to be elucidated.We aimed to investigate the feasibility of different echocardiographic methods to evaluate left ventricular systolic function in retrospective datasets, including left ventricular ejection fraction (LVEF), fractional shortening (FS), global longitudinal strain (GLS) and longitudinal strain (LS) as well as the incidence and timing of subclinical left ventricular dysfunction detected by these methods.2). In 68% of all 366 echocardiograms, LS analysis was feasible. Impaired LS values (<17%) was demonstrated in >40%, with concomitant impairment of either LVEF or FS in 20% and combined impairment of both LVEF and FS in <10%. Importantly, there were no cases of abnormal LVEF and FS without concomitant LS impairment.A retrospective longitudinal study was performed with re‐analysis of longitudinal echocardiographic data, acquired during treatment and early follow‐up, including 41 pediatric sarcoma patients, aged 2.1–17.8 years at diagnosis, treated at Astrid Lindgren Children's Hospital, Stockholm, Sweden, during the period 2010–2021. All patients had received treatment according to protocols including high cumulative doxorubicin equivalent doses (≥250 mg/mOur findings demonstrate feasibility of LS in a majority of echocardiograms and a high incidence of impaired LS during anthracycline treatment for childhood sarcoma. We propose inclusion of LS in pediatric echocardiographic surveillance protocols. The aims of the study were to explore the feasibility of different echocardiographic methods for evaluating left ventricular systolic function , as well as the incidence and timing of systolic dysfunction measured by these methods, during and early after anthracycline treatment.In this retrospective, single‐center, population‐based study, we have re‐evaluated cardiac function in serial echocardiograms performed during treatment and clinical follow‐up in childhood sarcoma patients treated according to protocols including high cumulative doxorubicin‐equivalent doses2 at Astrid Lindgren Children's Hospital, Stockholm, during the period 2010–2021. Patients meeting the inclusion criteria were identified through the Swedish Childhood Cancer Epidemiology Group register. Exclusion criteria were age >18 years at cancer diagnosis; cancer diagnosis other than sarcoma; treatment protocol without cumulative doxorubicin equivalent doses ≥250 mg/m2; and cardiac disease diagnosed before initiation of cancer therapy.The study inclusion criteria were age 0–18 years at sarcoma diagnosis and treatment according to protocols including cumulative doxorubicin equivalent doses ≥250 mg/mThe patient cohort included 41 patients  diagnosed with sarcoma; 24 with osteosarcoma, 15 with Ewing sarcoma and two patients with soft tissue sarcoma. Median age at the first echocardiogram  was 12.7 years (range 2.1–17.8 years).2) and to time after the last anthracycline treatment during follow‐up. The dose intervals correspond to the number of administered treatment cycles with anthracyclines; doxorubicin equivalent doses of 60, 75, and 80 mg/m2 were administered each treatment cycle for Ewing sarcoma, osteosarcoma and soft tissue sarcoma, respectively. The equivalence ratio of doxorubicin to other anthracyclines according to Feijen et alAll available echocardiograms were re‐analyzed, and medical reports were reviewed in all included patients. Patient outcomes and echocardiographic variables were longitudinally correlated to pre‐defined intervals of cumulative doxorubicin equivalent doses during treatment  at any time during treatment and vLVEF and FS after the end of treatment was evaluated at follow‐up within 6‐months and during the period of 1–2 years after the last anthracycline treatment. For dichotomised vLVEF and FS values, a 2 × 2 contingency table was set up and one‐sided Fisher's Exact Test was used to evaluate if decrease in vLVEF to values <53% and FS to values <0.28 were positively associated with abnormal LS (<17%) at any time during treatment at the 5% significance level. The null hypothesis was that a decrease in vLVEF to <53% and FS to <0.28 were independent of abnormal LS during treatment. For continuous vLVEF and FS values, the one‐sided Wilcoxon Test was used to test if the median values of vLVEF and FS were lower in the group of patients with abnormal LS during treatment at 5% significance level. The null hypothesis was that there would be no difference in the median values of vLVEF and FS between patients with abnormal LS (<17%) at any time during treatment and those with normal LS (≥17%) throughout treatment.33.1n = 24), Ewing sarcoma (n = 15), and soft tissue sarcoma (n = 2). The median age at the first echocardiogram was 12.7 years (range 2.1–17.8 years). Patients diagnosed with osteosarcoma and Ewing sarcoma received a median cumulative dose of doxorubicin equivalents of 450 mg/m2 , and 360 mg/m2 , respectively. Two patients with soft tissue sarcoma received doses of 240 and 320 mg/m2 doxorubicin equivalents (administered in cycles of 80 mg/m2). None of the patients received chest irradiation including the heart or the great vessels. The median follow‐up time was 32 months (range 6–97 months) after the first and 26 months (range 0–88 months) after the last anthracycline therapy. At the end of the study period, 30 of the 41 patients (73%) were alive. No patients were diagnosed with renal dysfunction (Glomerular Filtration Rate <60 mL/min/1.73 m2) or arterial hypertension, and there were no cardiac deaths during the entire study period. One patient with osteosarcoma was diagnosed with symptomatic heart failure of NYHA class III, 4.5 months after cessation of therapy with a cumulative doxorubicin dose of 450 mg/m2. Echocardiography at the time of clinical diagnosis of heart failure, revealed an LVEF of 15% , FS of 0.10 and LS of 5%. All the subsequent echocardiograms for this patient were excluded from the final analysis. At the presentation of heart failure, the biomarker N‐Terminal Pro–Brain Natriuretic Peptide was moderately elevated, 7900 ng/L , but high sensitivity cardiac Troponin‐T was normal. Prior to the presentation of symptomatic heart failure, six normal echocardiograms were performed during treatment and one echocardiogram 1 month after ending treatment. The latter, showed a normal FS (0.30) and a slightly reduced visual LVEF (50%). Unfortunately, neither LS nor biplane LVEF was available at this timepoint, due to poor quality of the stored echocardiographic images from apical views. No preceding risk factors such as arrhythmia, septicaemia, anemia, arterial hypertension, electrolyte disturbances, or renal failure were identified. With an LVEDD of +2.2 z‐scores, this was the only patient with significant dilatation of the left ventricle (LVEDD ≥ +2.0 z‐scores). A maximum LVEDD of +2.4 z‐scores was seen before improvement of cardiac systolic function occurred. Cardiac systolic function recovered completely  with standard heart failure treatmentThe patient cohort Table  included3.2In total, 366 echocardiograms were available for re‐analysis Figure . The ext3.3Of the 249 echocardiograms eligible for LS analysis . Besides LS, only one other systolic parameter (FS or vLVEF) was concomitantly affected in 22/105 (21%), while both FS and vLVEF were co‐affected in 8/105 (8%).Normal LS (≥17%), was registered in 144 echocardiograms (48%). In these echocardiograms, borderline values of FS (0.27) were detected in three and borderline vLVEF (50%) in one case, but there were none with abnormal values in both vLVEF and FS. The results of FS and vLVEF measurements were contradictory in 35 of all 366 echocardiograms. LS analysis was feasible in 22 of these 35 echocardiograms.2, but returned to normal values (≥17%) at 6 months follow‐up after ending treatment  during and after treatment. The median values for LS were abnormal (<17%) from cumulative doxorubicin equivalent doses of 180–225 mg/mt Figure .In a sub‐analysis, the cohort was dichotomised into patients with at least one echocardiogram with abnormal LS (<17%) and patients with normal LS (≥17%) throughout treatment Figure . In the 3.4The relationship between frame rate and heart rate (FR/HR‐ratio) as a measure for image quality may interfere with strain analysis.4Cardiac dysfunction before, during and after cancer treatment has important impact on both cancer and overall outcome, as well as long‐term quality of life, hence cardiotoxic cancer treatment regimens require serial evaluation of cardiac function by echocardiography.In adults, myocardial deformation, measured as GLS by 2D speckle‐tracking echocardiography, has become the preferred parameter for early detection of asymptomatic cancer therapy‐related cardiac dysfunction due to its high reproducibility and sensitivity,A good correlation between GLS and LS measurements has been demonstrated by others,2, indicating myocardial injury at lower doses than considered to be “high risk”.Longitudinal strain was the most frequently affected parameter of systolic function . This finding, together with results from previous studies of childhood cancer survivors, indicates a superior sensitivity of LS compared to LVEF and FS.The methodological limitations of standard echocardiographic surveillance of pediatric cancer patients, including only FS and LVEF, are demonstrated in this study. Although FS had excellent feasibility, as available in 98% (357/366) of the echocardiograms, there was only one case with impaired FS (0.27), as the only affected systolic parameter. In contrast, LVEF measurements by the recommended biplane method of disk summationBesides the size of the patient cohort and sort follow‐up time, a weakness of this study, shared by other retrospective studies on echocardiograms performed in clinical practice, was the limitation of acquired images.5Despite the limitations of cohort size and follow‐up time, our results demonstrate a high feasibility of LS also in retrospective echocardiographic datasets. Our results indicate that LS entails higher sensitivity for detecting subclinical changes in cardiac function compared to FS and LVEF. In conjunction with previous studies,To improve the understanding of the variable course of individual patients and to provide outcome measures for further research on early detection of cardiovascular toxicity, adherence to detailed standardized echocardiographic protocols including left ventricular systolic strain analysisConceptualization: Maria Sjöborg Alpman, Agneta Månsson Broberg, Nikolas Herold, Aristomenis Manouras, and Jan‐Inge Henter. Methodology: Maria Sjöborg Alpman, Agneta Månsson Broberg, Nikolas Herold, and Jan‐Inge Henter. Validation: Maria Sjöborg Alpman, Annica Jarting, and Kerstin Magnusson. Investigation: Maria Sjöborg Alpman, Annica Jarting, and Kerstin Magnusson. Formal Analysis: Maria Sjöborg Alpman, Agneta Månsson Broberg, and Nikolas Herold. Resources: Jan‐Inge Henter, Agneta Månsson Broberg, and Nikolas Herold. Writing—Original Draft: Maria Sjöborg Alpman, Agneta Månsson Broberg, and Nikolas Herold. Writing—Review and Editing: Maria Sjöborg Alpman, Agneta Månsson Broberg, Nikolas Herold, Aristomenis Manouras, Jan‐Inge Henter, Annica Jarting, and Kerstin Magnusson. Visualization: Maria Sjöborg Alpman. Supervision: Jan‐Inge Henter. Funding Acquisition: Jan‐Inge Henter.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. The authors have stated explicitly that there are no conflicts of interest in connection with this article.Ethical approval was received from the Swedish Ethical Review Board (Dnr 2020‐02385 and Dnr 2021‐06061‐02).Table S1. Longitudinal echocardiographic characteristics of patients with abnormal LS (<17%) at any time during anthracycline treatment and normal LS (≥17%) at all times during treatment, with respect to LS, vLVEF and FS during follow‐up within 6 months and at 1–2 years after end of anthracycline treatment. Statistical analysis was performed to evaluate the association between abnormal LS (<17%) at any time during treatment and vLVEF and FS at follow‐up within 6 months and during the period 1–2 years after the last anthracycline treatment. Fisher's exact test and Wilcoxon test returned statistically non‐significant results. FS, fractional shortening; IQR, interquartile range; LS, longitudinal strain; n, numbers; NA, not available; vLVEF, visual left ventricular ejection fraction; yr, year.Figure S1. (A) and (B) Echocardiograms with normal LS (≥17%) as well as echocardiograms with abnormal LS (<17%) demonstrate a FR/HR‐ratio ≥0.7 in a majority of cases: FR, frame rate; HR, heart rate; LS, longitudinal strain.Click here for additional data file.