To investigate the protective effect of sevoflurane preconditioning on renal ischemia‐reperfusion injury  and its related mechanism.Eighty healthy adult male SD rats were randomly divided into control group (Sham group), model group (RIRI group), sevoflurane pretreatment group (Sev group) and TRPM7 inhibitor combined with sevoflurane pretreatment group (T + Sev group), 20 animals in each group. Hematoxylin‐eosin (HE) staining was used to observe the pathological changes of renal tissue, and the levels of creatinine and urea nitrogen in each group were detected. Deoxyribonucleic acid terminal transferase‐mediated dUTP nick end labeling (TUNEL) assay was used to detect renal cell apoptosis, and Western blottingwas used to detect the expression of apoptotic proteins cleaved‐caspase‐3, bax, Bcl‐2, and TRPM7 in renal tissue; Detection of oxidative stress‐related index levels in renal tissue and levels of inflammatory factors in renal tissue and serum.p < .05); The scores, apoptosis rate, MDA level, and relative expression of inflammatory factor levels were decreased, and SOD levels were increased . Compared with the Sev group, the renal tissue pathological damage in the T + Sev group was aggravated, creatinine, blood urea nitrogen levels increased, apoptosis increased, apoptosis‐related proteins cleaved‐caspase‐3, bax, Bcl‐2 showed increased apoptosis, malondialdehyde (MDA) levels, inflammatory factor levels increased, ultrahigh The levels of oxide dismutase (SOD) were decreased .Compared with the Sham group, the renal tissue pathological damage was aggravated, the levels of creatinine and blood urea nitrogen were increased, and the apoptosis was increased in the RIR group and the Sev group. Death, malondialdehyde (MDA) levels and inflammatory factors were increased, and superoxide dismutase (SOD) levels were decreased (all Therefore, we believe that sevoflurane is involved in the protection of rat renal ischemia‐reperfusion injury by downregulating the expression of TRPM7. We found that sevoflurane participates in the protection of rat renal ischemia‐reperfusion injury by downregulating the expression of TRPM7, which provides a basis for further clinical trials of sevoflurane in renal ischemia‐reperfusion injury protection new direction. It also has kinase activity and can mediate intracellular and extracellular signal transduction. Various biopathophysiological processes of cells.22.1Eighty healthy male adult SD rats, 6–8‐week‐old, weighing 220–320 g, were provided by Experimental Animal Center of Jiangsu University. All experimental animals were kept in a suitable ventilated environment , and all animals were free to drink tap water and feed. Feed was provided by Jiangsu Synergetic Pharmaceutical Bioengineering Co., Ltd. SD rats were randomly divided into control group (Sham group), model group (RIRI group), sevoflurane pretreatment group (Sev group), and TRPM7 inhibitor group (Sev + T group), 20 rats in each group. Rats in the Sham group were treated with sham surgery. The renal ischemia‐reperfusion model was established in the RIRI group. Rats in the Sev group were intervened with sevoflurane 1 h before the establishment of the pulmonary IRI model. TAK‐242 (10 mg/kg) was injected into the tail vein of the rats in the T + Sev group, followed by sevoflurane intervention 1 min later, and a renal RIRI model was established 1 h later. This study was approved by the Ethics Committee of Changzhou Hospital of Traditional Chinese Medicine .2.2Sevoflurane was purchased from Wuhan Aimeijie Technology Co., Ltd.; 10% chloral hydrate was purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd. Deoxyribonucleic acid terminal transferase‐mediated dUTP nick‐end labeling  staining The kit and enzyme‐linked immunosorbent assay (ELISA) kit were purchased from Beijing Soleibao Technology Co., Ltd.; cleaved‐caspase‐3, bax, Bcl‐2, TRPM7 antibody, β‐actin antibody, and secondary antibody were all Purchased from Abcam; fully automatic biochemical analyzer was purchased from Berkam.2.3Experimental rats were injected intraperitoneally with 10% chloral hydrate 0.3 ml/100 g. After the depth of anesthesia reached the surgical requirements, the rats were dehaired and placed under a warm light on the operating table. The skin, rectus abdominis and peritoneum were incised layer by layer along the midline of the upper abdomen, the left and right kidneys were exposed, the renal pedicle was freed, the renal pedicle was clamped to block the renal blood flow for 45 min, and then released to allow the blood flow to recanalize, and the color change of the kidney was observed. The kidneys changed from dark purple to red within 5 min, indicating that the ischemia‐reperfusion injury model was successfully established.33.1Lung tissues fixed in 4% paraformaldehyde solution for 24 h were taken to make paraffin sections and stained with hematoxylin‐eosin (HE). After drying, the sections were dewaxed with xylene, washed with ethanol gradient, stained with hematoxylin for 5 min, differentiated with hydrochloric acid and ethanol, stained with eosin for 1 min, rinsed with tap water, dehydrated with ethanol gradient, transparent with xylene, and mounted with neutral resin. The pathological changes of renal tissue were observed by light microscope.3.2TUNEL apoptosis detection kit was used to detect the apoptosis of renal tissue cells and calculate the apoptosis rate. The kidney tissue paraffin sections were deparaffinized and rehydrated, digested with proteinase K, incubated in the dark after adding TUNEL reaction buffer, 4ʹ,6‐diamidino‐2‐phenylindole  and mounted with antifluorescence quencher. The apoptosis of each group was observed under a fluorescence microscope, and the positive cells showed red fluorescence. Five slices were selected in each group, and five fields of view (×200) were randomly selected for each slice.3.3Partially cryopreserved lung tissue was taken, ground in liquid nitrogen, and fully lysed by adding RIPA protein lysing solution to extract total protein. The protein concentration was determined by BCA method. The protein samples were electrophoresed through a polyacrylamide gel, and then blocked with 5% skim milk at room temperature for 1.5 h after transfer. Primary antibodies  were incubated at 4°C overnight, and the secondary antibody (11000) was incubated at room temperature for 1 h, and ECL substrate was added dropwise for luminescence and color development. The grayscale of the bands was detected by a gel image analysis system, and the relative expression level of the target protein was calculated.3.4Part of the cryopreserved lung tissue was taken, homogenized by adding nine times normal saline, and the supernatant was collected after centrifugation for detection. The level of malondialdehyde (MDA) was detected by thiobarbituric acid colorimetric method, and the level of total superoxide dismutase (SOD) was determined by WST‐8 method.3.5The levels of interleukin (IL)‐6 and tumor necrosis factor (TNF)‐α in serum were detected by ELISA. The level of myeloperoxidase (MPO) in lung tissue was determined by dianisidine colorimetric method.3.6p < .05 means the difference is statistically significant.Statistical analysis was performed using SPSS 19.0 software. The measurement data conforming to the normal distribution were expressed as mean ± standard deviation, the comparison between multiple groups was performed by one‐way analysis of variance, and the comparison between groups was performed by LSD‐t test. 44.1p < .05). Compared with the RIRI group, the renal pathological damage score of the rats in the Sev group was decreased (p < .05)  and so forth. The Sev group significantly improved RIR‐induced histological changes Figure . Compare) Figure .4.2p < .05). Compared with the RIRI group, the serum creatinine of the Sev group was decreased (p < .05) . Compared with the RIRI group, the serum urea nitrogen in the Sev group was decreased (p < .05)  Figure .4.3p < .05)   Figure .p < .05), and the expression of Bcl‐2 was decreased (p < .05). Compared with the RIRI group, the expressions of apoptosis‐related proteins cleaved‐caspase‐3 and bax in the kidney tissue of the Sev group were lower than those of the RIRI group (p < .05), and the expression of Bcl‐2 was higher than that of the RIRI group (p < .05). The apoptosis of kidney tissue of rats in RIRI group and Sev group increased, and sevoflurane could improve the apoptosis of renal cells caused by renal ischemia‐reperfusion . Compared with the RIRI group, the cellular TRPM7 protein concentration in the renal tissue of the Sev group was decreased (p < .05) . The expression of TRPM7 on renal tubular epithelial cells was decreased (p < .05)  and so forth. The Sev group significantly improved the histological changes caused by RIR, and after inhibiting TRPM7, the protective effect of sevoflurane on renal tissue was significantly reduced Figure .4.7p < .05). Compared with the RIRI group, the serum creatinine of the Sev group was decreased (p < .05). After inhibiting TRPM7, the effect of sevoflurane on reducing the rat creatinine was significantly reduced (p < .05). . Compared with the RIRI group, the serum urea nitrogen in the Sev group was lower, and the effect of sevoflurane on reducing the urea nitrogen in the rats was significantly reduced after inhibiting TRPM7. (p < .05)  Figure .4.8p < .05). Compared with the RIRI group, the apoptosis rate of renal tissue in the Sev group was lower than that in the RIRI group (p < .05). After inhibiting TRPM7, the effect of sevoflurane on reducing the apoptosis of the rats was significantly reduced (p < .05) , and the expression of Bcl‐2 was decreased (p < .05). p < .05). Compared with the RIRI group, the expressions of apoptosis‐related proteins cleaved‐caspase‐3 and bax in the kidney tissue of the Sev group were lower than those of the RIRI group (p < .05), and the expression of Bcl‐2 was higher than that of the RIRI group (p < .05). The apoptosis of kidney tissue of rats in RIRI group and Sev group increased. After inhibiting TRPM7, the effect of sevoflurane on reducing apoptosis in rats was significantly reduced (p < .05)  Figure . Sevoflu4.9p < .05). The expressions of oxidative stress‐related indexes SOD and MDA in the serum of the Sev group were lower than those of the RIRI group (p < .05). After inhibiting TRPM7, the effect of sevoflurane on reducing the serum oxidative stress level of the rats was significantly reduced (p < .05). Compared with the Sham group, the expressions of inflammatory factors IL‐6, TNF‐α and MPO in the serum of the RIRI group, Sev group and Sev + T group were increased (p < .05). The expressions of oxidative stress‐related indicators IL‐6, TNF‐α, and MPO in the serum of the rats in the Sev group were lower than those in the RIRI group (p < .05). After inhibiting TRPM7, the effect of sevoflurane on reducing the levels of serum inflammatory factors in rats Significantly decreased (p < .05) (Figure Compared with Sham group, the expressions of oxidative stress‐related indexes SOD and MDA in serum of rats in RIRI group, Sev group and Sev + T group were increased  Figure . In conc5Compared with other inhaled anesthetics, sevoflurane has the advantages of rapid induction, less respiratory irritation, low solubility, better hemodynamic stability, rapid absorption and clearance, and rapid and complete recovery. The advantage is that it is a relatively safe and ideal inhalation anesthetic.In recent years, studies have found that sevoflurane can reduce the ischemia‐reperfusion injury of the heart, brain, lung, liver, and other organs. In this study, an acute renal ischemia‐reperfusion injury model was constructed through animal experiments, renal function was detected, and pathological changes in renal tissue were observed to explore the protective effect and possible mechanism of sevoflurane on renal ischemia‐reperfusion injury. The experimental results show that: sevoflurane regulates the expression of TRPM7 protein, controls the process of apoptosis, further significantly reduces serum creatinine and blood urea nitrogen levels, and increases SOD activity, which can play a role in resisting acute renal ischemia‐reperfusion injury. At present, there is still no effective method to prevent and treat renal ischemia‐reperfusion injury in clinical practice. Therefore, it is an urgent clinical need to deeply explore the pathogenesis of sevoflurane and RIRI, so as to find an effective method to prevent and treat the degree of RIRI. This study was modeled according to the previous literature on ischemia reperfusion for the study of this experiment.Inflammatory response is a key factor in the development of RIRI.The inflammatory response regulation mechanism of renal RIRI process is relatively complex, and TRPM7, as an inflammatory switch, can activate the body's immune inflammatory response by regulating the RP‐1/NAD + pathway to regulate the necroptosis signaling pathway.In addition, this study has many shortcomings. First of all, relevant in vitro experiments have not been carried out for further verification. Second, the specific molecular mechanism that sevoflurane regulates the expression of TRPM7 and participates in the protection of rat renal ischemia‐reperfusion injury has not yet been explored. These will be further improved in subsequent experiments.In summary, through this experimental study, we found that sevoflurane participates in the protection of rat renal ischemia‐reperfusion injury by downregulating the expression of TRPM7, which provides a basis for further clinical trials of sevoflurane in renal ischemia‐reperfusion injury protection new direction.Xudong Xu and Rongrong Deng conceived the study and designed the experiments. Rongrong Deng, Lu Zou, and Xiaoyan Pan contributed to the data collection, Zhifeng Sheng, Da Xu, and Tingting Gan performed the data analysis and interpreted the results. Xudong Xu wrote the manuscript; Xudong Xu and Rongrong Deng contributed to the critical revision of article. All authors read and approved the final manuscript.The authors declare no conflict of interest.


Approximately 18% of patients with atrial fibrillation (AF) undergo a repeat ablation within 12 months after their index ablation. Despite the high prevalence, comparative studies on nonpulmonary vein (PV) target strategies in repeat AF ablation are scarce. Here, we describe 12 months efficacy of non‐PV and PV target ablations as a repeat ablation strategy.n = 140) included PV reisolation, posterior wall isolation, mitral line, roofline, and/or complex fractionated atrial electrogram ablation. PV target ablation (n = 140), included reisolation and/or wide atrium circumferential ablation. Patients' demographics and rhythm outcomes during 12 months follow‐up were analyzed.A multicentre retrospective, descriptive study was conducted with data of 280 patients who underwent repeat AF ablation. The ablation strategy for repeat ablation was at the operators' discretion. Non‐PV target ablation (p = .001). Similarly, a significantly higher AF and atrial tachycardia (AT) recurrence rate was observed after non‐PV target ablation compared to PV target ablation (36.4% vs. 22.1% and 22.9% vs. 10.7%). After adjustment, a significantly higher risk of AT recurrence remained in the non‐PV target group. Both groups significantly de‐escalated antiarrhythmic drug use; de‐escalation was more profound after PV target ablation. Patients with isolated PVs during non‐PV target ablation had a significantly higher risk for AF recurrence than those with reconnected PVs.At 12 months, more atrial tachyarrhythmias were observed in the non‐PV target group (48.6%) compared to the PV target group (29.3%, Compared to PV target ablation, non‐PV target repeat ablation did not improve outcomes after 12 months and was independently associated with an increased risk for AT recurrences. Eligible patients had a previous AF (index) ablation and the primary indication for the repeat ablation was AF. Cases were performed with conventional RF or cryoballoon ablation for both the initial and repeat ablations. We excluded patients in whom AT was the primary indication for repeat ablation and patients who withdrew consent to use their data.2.1This study was conducted in full accordance with the Declaration of Helsinki  and the OLVG local ethics committee issued a waiver for an informed consent form. For study inclusion, a consent form to use clinical data was sent to all patients eligible for this study. Patients were asked to sign and return the consent form by mail (opt‐in). For patients who did not respond to our initial request, we tried to contact them by telephone. If all those attempts failed, data of the patients were included in the analyses following the Dutch Medical Treatment and Agreement Act.2.2We aimed to compare the clinical outcome of non‐PV target versus PV target ablation strategies during the first repeat AF ablation. Non‐PV target ablation was defined as PV reisolation in all patients with PV reconnection + additional LA ablation, including posterior wall isolation, mitral lines, roofline, and/or ablation of complex fractionated atrial electrograms (CFAEs). AF trigger ablation was not performed in this study. PV target ablation included reisolation of PV with or without wide antrum circumferential ablation or wide antrum circumferential ablation in case of already isolated PVs.The primary outcome was the reported freedom of any atrial tachyarrhythmia at 12 months after the repeat AF ablation.2.3The repeat AF ablation strategy and modality was at the operators' discretion and reflected the standard of care. Ablation modalities for repeat ablation included conventional RF  or the second‐generation cryoballoon (Medtronic). PV reisolation was defined as focal touch‐up ablation of reconnected PVs until entry and exit blocks were confirmed. Wide antrum circumferential ablation in patients with isolated or reconnected PVs increased the lesion size around the PV ostia and was considered a PV target strategy. Posterior wall isolation consisted of a roofline between the left and right superior PVs and a posterior line between inferior PVs. A mitral line was defined as any ablation line between the mitral valve annulus and a conduction barrier such as left inferior PV, right superior PV, or posterior ablation line. CFAE ablation was defined as ablation of fractionated electrograms with two or more deflections, continuous deflection of a prolonged electrogram, and/or electrograms with an average cycle length <120 ms over a 10‐s period.2.4Patients underwent routine clinical care follow‐up via referring hospitals. All available rhythm recordings during the 12 months follow‐up were retrieved from the medical records and analyzed for the current analysis. Recurrence of any atrial tachyarrhythmia was defined as an AF or AT episode lasting >30 s and documented on an electrocardiographic recording, including electrograms retrieved from cardiac implantable electronic devices.2.5For a clarification purpose, we categorized the AAD use data as Grade 1—patients who used beta‐blockers or calcium antagonists (Vaughan Williams Class II or Class IV AAD) or no ADD, Grade 2—patients who used Class I AAD or sotalol, and Grade 3—patients who used amiodarone. De‐escalation of AAD use was defined as the discontinuation of AAD or the use of a lower grade AAD than before the procedure.2.6LA dimensions and left ventricle (LV) function were interpreted as normal, mildly, moderately, or severely enlarged/impaired as per guideline recommendations.2.7t test, nonparametric test, and χ2 test were performed for comparative analyses between groups. Multivariate logistic regression analysis was performed to adjust for confounding baseline characteristics. Covariates were included in the multivariate analysis if they changed the exposure coefficient by >10%.Normally distributed variables are presented as mean ± standard deviation and non‐normal data as the median and interquartile range. Parametric 33.1Two hundred and eighty‐six patients undergoing repeat AF ablation were eligible for this study, of whom 280 (98%) were included. Four patients were excluded because they refused consent, and two other patients died before the end of the 90‐day blanking period. The baseline clinical characteristics are summarized in Table p < .001), had a higher CHA2DS2VASc , diabetes, stroke (doubled), vascular disease, age (≥ 65), sex) score , and a higher incidence of severe LA enlargement  than those in the PV target group. Persistent AF was more frequent in the non‐PV target group compared to the PV target group  (Table p = .011) (Table Non‐PV target and PV target ablation were both performed in 140 (50%) patients. Patients undergoing non‐PV target ablation were older , whereas only five patients (1.8%) were treated with cryoballoon, all in the PV target group. Contact force‐sensing catheters were used in 112 (80.0%) patients and 108 patients (77.1%) in the non‐PV target and PV target group, respectively. Electrophysiological mapping before repeat ablation revealed that all PVs were isolated in 72 (25.7%) patients  , mitral line , roofline , and CFAE ablation   and wide atrium circumferential ablation  (Table p = .111). Cavotricuspid isthmus ablation was performed in 77 patients (27.5%), and accounted for 29.3% of non‐PV target and 25.7% of PV target group, p = .592.Repeat ablation was predominantly performed with conventional RF n = 75, 98.2%%) Table . Complet%) Table . An AT w3.3p = .600), including vascular groin complication , cardiac tamponade , exacerbation of heart failure , pericarditis , hematuria after urinary catheter placement , and complete heart block .Sixteen (5.7%) procedural complications occurred in 15 patients  (Table p = .154)  . The cryoballoon ablation modality in the first ablation was the only covariate included in the multivariate model of which effect remained statistically significant after adjustment   patients at 12 months follow‐up, more frequently in the non‐PV target  Figure . AF recu) Figure . In tota) Figure .3.5n = 135, PV target n = 132) and demonstrated outcomes on any atrial tachyarrhythmia, AT, and AF recurrence in line with the multivariate analysis presented in Figure p = .074) and had a statistically significant higher risk for AT recurrence . There was no statistically significant difference in AF recurrence risk between patients who underwent non‐PV target ablation and patients who underwent PV target ablation .Logistic regression with inverse propensity weighting was performed in 267 patients (non‐PV target 3.6p = .864) or AF  recurrences between the two ablation strategies  after adjustment for chronic obstructive pulmonary disease and the use of contact force‐sensing catheters. LA size and body mass index were excluded from the model that resulted in a lower AIC  patients, one or more PV reconnections were identified during electrophysiological mapping at the repeat AF ablation procedure. Of these, 69 and 139 patients underwent non‐PV target and PV target ablation, respectively. After adjustment for confounding covariates, there was no significant difference in the number of reported atrial tachyarrhythmias . We performed subanalyses to determine the arrhythmia recurrence risk of patients with isolated PVs before repeat AF ablation who underwent non‐PV target ablation compared to patients with reconnected PVs who underwent non‐PV target ablation   after adjustment for cryoballoon ablation during index procedure, non‐vitamin K oral anticoagulants or calcium antagonist use, and posterior wall isolation  than patients with PV reconnections 33.2%) (% (p = .0) Figure . Addition Figure . To avois Figure .3.8p < .001; Figure p = .003; Figure p = .167; Figure p = .130; Figure p = .066; Figure A significant de‐escalation of AAD therapy (defined as discontinuation of AAD or use of lower grades of AAD) was observed in both groups (3.9p < .001). This improvement was more evident in patients who underwent PV target ablation , in comparison with patients who underwent non‐PV target ablation  (group comparison p = .003). Similar findings were observed in patients with reconnected PVs before repeat AF ablation, both groups improved significantly in EHRA class (p < .001) and the improvement was more evident in patients who underwent PV target ablation (group comparison p = .014). Also, patients with PV reconnection at repeat ablation undergoing non‐PV target ablation demonstrated a greater improvement in the EHRA score , in comparison with patients with isolated PVs  (group comparison p < .001).EHRA scores improved from 2.47 ± 0.62 before repeat ablation to 1.66 ± 0.77 after 12 months follow‐up (4In this multicentre retrospective study, patients suffered significantly higher recurrences of atrial tachyarrhythmia, AF, and AT after non‐PV target ablation compared to those undergoing PV target ablation. After adjustment for covariates, the risk for atrial arrhythmia or AF recurrences was not statistically significant anymore. Hence, the risk of atrial tachyarrhythmia recurrence was strongly driven by baseline characteristics. However, AT recurrence risk for patients after non‐PV target ablation remained statistically significant. In addition, most patients were able to step down in level of AAD after repeat ablation, but not those with isolated PVs and non‐PV targeted ablation. Patients in both groups showed significant improvement of AF‐related symptoms, more evident in the PV target group.4.1Freedom of atrial tachyarrhythmias after repeat ablation ranges between 50% and 75% depending on patient characteristics, ablation strategy, and the presence of reconnected PVs.4.2In our study, patients with reconnected PVs at repeat non‐PV target ablation had better outcomes compared to patients with isolated PVs, suggesting that occurrence and maintenance of atrial tachyarrhythmias are PV‐dependent in many patients. In an observational cohort study for 143 subjects with first repeat AF ablation, patients with reconnected PVs similarly showed a better atrial arrhythmia‐free survival than patients with isolated PVs.In the RACE‐AF trial, all patients underwent a repeat procedure 4–6 months after the first AF ablation.4.3Most of the non‐PV target ablation strategies data are derived from the first ablation procedures. We believe that the findings of the studies during the first AF ablation discussed below support our data in the setting of repeat AF ablation. In our study, 90 patients in the non‐PV target ablation group underwent posterior wall isolation. A previously meta‐regression study by Sau et al.Magnetic resonance imaging‐identified LA tissue fibrosis has been associated with more atrial arrhythmia recurrence.Taken together, there is no striking evidence in the literature that additional non‐PV target ablation during first or repeat AF ablation improves outcomes compared to PV target ablation alone. Supported by these data, we believe that the role of the PVs is crucial, and extensive LA ablation should be used only in a selected patient with extra caution. The mechanism of AF beyond the PVs is complex, warranting further investigation to confirm the efficacy of tailored AF ablation approaches in patients with isolated PVs. Besides, a remaining question is what a realistic therapeutic goal is in patients with extensive fibrosis or isolated PVs.4.4This study has some limitations. First, only patients were included who underwent repeat AF ablation, and the admission to that therapy was based on a shared decision between patient and operator. Therefore, patients who did not undergo repeat AF ablation despite having an atrial tachyarrhythmia recurrence after a first ablation procedure were excluded, and our results are representative only for patients actually undergoing repeat ablation. Second, the ablation strategy was performed at the operators' discretion and differences in baseline characteristics were observed between both ablation strategies. Therefore, adjustment was performed for covariates that changed the exposure coefficient to >10%. Thus, baseline characteristics that were associated with an ablation strategy were included in the multivariate model. Also, a sensitivity analysis was performed with inverse propensity weighting to assess the robustness of the multivariate model and demonstrate comparable outcomes. However, we were unable to correct for electrophysiological findings, as the electroanatomical mapping data were unavailable. Nevertheless, the notable differences in atrial tachyarrhythmia recurrence between groups provide important insights for daily practice. The actual efficacy of non‐PV target ablation should be investigated in randomized trials. It is also important to reconsider acceptable outcomes in terms of arrhythmia recurrences and patient satisfaction in patients undergoing non‐PV target ablation. The current consensus of the absence of >30 s atrial tachyarrhythmia is likely very strict and ignores symptomatic improvement. Third, the non‐PV targets strategies were a “mixed bag” of LA ablation and this study was underpowered to determine the effect of each ablation site. Fourth, none of the patients included in this study underwent AF trigger ablation. Another limitation of this study is that routine follow‐up instead of a structured study follow‐up scheme was used. Nonetheless, follow‐up visits and monitoring were the same in both groups.5In patients undergoing first repeat AF ablation, non‐PV target ablation failed to improve arrhythmia free survival compared to PV target approaches. Atrial tachyarrhythmia, AF, and AT recurrences were more frequently observed in patients undergoing non‐PV target ablation. These differences were partially driven by the baseline characteristics of patients. However, patients with a non‐PV target ablation remained at higher risk for AT recurrence after adjustment for differences in baseline characteristics. Therefore, randomized controlled trials are needed to determine the actual effect of non‐PV target ablation during repeat AF ablations.Daniel Mol reports speaker/consultancy fee from Abbott. Giovanni J. M. Tahapary reports speaker fees from Medtronic. Joris R. de Groot reports research grants to his institution from Abbott, Atricure Inc., Bayer, Boston Scientific, Daiichi Sankyo, Johnson & Johnson, Medtronic, consultancy/speaker fees from Abbott, AtriAN Medical, Atricure Inc., Bayer, Biotronik, CVOI, Daiichi Sankyo, IPP Med, Itreas, Medtronic, Novartis, Servier, outside the submitted work. Jonas S. S. G. de Jong reports consultancy fees from Medtronic and speaker fees from Daiichi‐Sankyo and Bayer, outside the submitted work.Supporting information.Click here for additional data file.


It is uncertain whether depression might affect cognitive function in Alzheimer’s disease (AD). Most of studies on the effect of depression treatment on cognitive function in AD were briefly evaluated by Mini-Mental State Examination (MMSE). MMSE is poor sensitive to detect cognitive change. This study examined the cognitive response to depression treatment in AD via multi-domain assessment. In addition, we explored whether effect of depression treatment in AD is different those of late-life depression (LLD).t test was performed to compare the degree of change between the groups. Paired t tests were used to determine cognitive function changes in each depression treatment responder group.This study include AD patients with depression (AD + D) and without depression (AD − D), LLD patients (LLD), and healthy controls (HC). The patients were treated according to their diagnosis for 16 weeks: acetylcholinesterase inhibitors (AChEIs) and selective serotonin reuptake inhibitors (SSRIs) for AD + D, AChEIs for AD − D, and SSRIs for LLD. The cognitive changes from pre- to post-treatment were compared between AD + D and AD − D or LLD and HC. An independent sample At baseline, AD + D had more impairment in language function compared to AD − D, and LLD had greater deficit in executive function than HC. After depression treatment, more impaired cognitive domains at baseline were improved in AD + D and LLD, respectively. Moreover, AD + D showed an improvement in the global cognitive function (MMSE).Results indicated that language function was influenced by depression in AD, which is first evidence for specific cognitive domain related to depression in AD. Our finding indicates that depression could negatively impact cognitive function, and depression treatment may have beneficial cognitive effect in both AD and LLD. This study suggests the importance of early detection and treatment of depression in AD and LLD.Trial registration Clinical Research Information Service, CRIS, ID#: KCT0004041, Registered 5 June 2019, retrospectively registered after first patient enrollment date (4 March 2014) https://cris.nih.go.kr/cris/search/detailSearch.do?seq=14140&status=5&seq_group=14140&search_page=M. Depression is one of the most common comorbidities in patients with Alzheimer's Disease (AD). It has been estimated that 10–20% of AD patients have major depression, and an additional 40–50% of patients experience depressive symptoms , worse qSeveral cross-sectional comparisons indicate that AD patients with depression (AD + D) have more cognitive impairment than AD patients without depression (AD − D) , 66. In A clinical review of the literature and meta-analyses several studies, which have evaluated the impact of antidepressant treatment on cognitive function among AD patients with depression, suggested conflicting results , 47.Some studies have shown an improvement in global cognitive function after use of serotonin reuptake inhibitors  or tricyclic antidepressants  medication in AD patients with depression , 63. HowIn contrast, several studies have reported that cognitive function was unchanged despite a reduction in depressive symptoms or recovery after SSRIs  , 43 or TUnlike depression in AD, it has been widely suggested that late-life depression (LLD) without dementia could induce cognitive impairment in the elderly , 67, 69.As mentioned above, in most previous studies, cognitive response to depression treatment in AD was crudely measured by the MMSE, which showed conflicting results. To evaluate the influence of depression treatment on cognitive function more sensitively, diverse cognitive domains must be investigated using a comprehensive neuropsychological test battery.Therefore, we investigated cognitive alterations between AD + D and AD − D via multi-domain assessment, including language function, memory, constructional praxis, and executive function, as well as global cognitive function in the MMSE, along with depression treatment. The effects of antidepressant treatment on each cognitive domain were also examined. In addition, we examined whether the effect of depression treatment in AD is different from that in LLD.Specifically, this study examined the following by multi-domain assessment: (i) whether cognitive function is different according to the presence or absence of depression in AD group or elderly without dementia group and (ii) whether cognitive function changes after depression treatment in AD + D group or LLD group, and, if so, which cognitive domains are related to depression.The participants were recruited from the Dementia Clinic of Chuncheon Sacred Heart Hospital. The inclusion criteria for the AD patient group (AD + D or AD − D) were as follows: (a) diagnosis of AD: met the criteria for AD via the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) ; and neuMoreover, depression in the elderly without dementia was assessed according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition DSM-5) [ [5].The patients who met any of the following criteria at screening were excluded: (a) a history of taking antidepressants within 4 weeks; (b) treatment with memantine medication for at least 4 weeks; and (c) Parkinson's disease, stroke, brain tumor, or normal brain pressure hydrocephalus.Eligible participants were enrolled 2 years (March 2014 to June 2016). A total of 95 participants were included in this study. Participants consisted of patients with newly diagnosed AD and LLD, as well as healthy controls with non-demented and non-depressed volunteers: 29 AD patients with depression (AD + D), 36 AD patients without depression (AD − D), 15 patients with late-life depression (LLD), and 15 healthy controls (HC) were included in the final sample.All participants and their caregivers provided written informed consent. The Ethics and Medical Research Committee of Chuncheon Sacred Heart Hospital approved the study protocol.All participants were scheduled at baseline and at 4, 8, and 16 weeks. Depressive symptoms were rated at every visit, while cognitive function, overall functioning, and other psychosocial factors were examined at the baseline and final visits.Clinical groups received treatment with Acetylcholinesterase inhibitors  (for AD) and/or selective serotonin reuptake inhibitors  (for depression) according to diagnosis during the 16 weeks: AChEIs and SSRIs for AD + D, AChEI for AD − D, and SSRIs for LLD.Depressive symptoms in AD + D and LLD were objectively assessed by the Cornell Scale for Depression in Dementia (CSDD)  and the Cognitive function was assessed using several subtests from CERAD-K neuropsychological assessments and the Seoul Neuropsychological Screening Battery (SNSB) . The neuActivities of Daily Living (ADL) were measured using the Seoul Instrumental Activities of Daily Living (S-IADL)  for AD pt test was performed to compare the degree of change between the groups. Paired t tests were used to determine cognitive function changes in each treatment responder group. Cohen’s d (for a t test) and/or partial eta squared (ηp2) (for an ANCOVA) were reported as a measure of effect size. The statistical analysis was performed usi148ng SPSS Statistics 23.0.For the descriptive statistics, a Fisher's exact test was used. To assess differences in cognitive function between groups at baseline, analysis of covariance (ANCOVA) with covariates of age, sex, and the duration of education was conducted. An independent sample p = 0.006).Table F = 5.346, p = 0.024, ηp2 = 0.082). The mean score of MMSE-KC, which measures global cognitive function, in LLD was lower than that of HC . The LLD group had lower mean scores of Contrasting, Go/No-Go, and Semantic Fluency tests, which are executive function tests, compared to HC group .Table t = 3.401, p = 0.001, Cohen’s d = 0.86), SIS , and Suicidality  assessments and lower mean scores on the WHO-5  compared to AD − D. The LLD group showed higher mean scores on the S-IADL  and Suicidality  and lower mean scores on the WHO-5  compared to the HC group.There were also significant differences in other factors between the depressed and the non-depressed, in both the AD and the elderly without dementia groups. AD + D had higher mean scores on the NPI . Figure t = 5.684, p = 0.000, Cohen’s d = 1.340 and t = 8.215, p = 0.000, Cohen’s d = 2.279, respectively).To determine the effect of depression treatment on clinical factors, we first analyzed whether depressive symptoms were significantly reduced after 16 weeks in AD + D and LLD subgroups with depression. In both AD + D and LLD, the mean score of depressive symptoms (as measured by CSDD for AD + D or HAMD for LLD) within each subgroup was significantly diminished from baseline to final . The LLD group showed a significant improvement on the Go/No-Go test as compared to the HC group .Table The response rate of depression treatment was 62.07% (18 of 29) in AD + D (defined as a ≥ 50% reduction in CSDD score) and 86.67% (13 of 15) in LLD (defined as a ≥ 50% reduction in HAMD score). At 16 weeks, we examined the changes in cognitive function before and after treatment according to the response to depression treatment to confirm whether the improvements in cognitive function in some domains were indeed due to the treatment effect of the medication.t = −2.317, p = 0.033, Cohen’s d = −0.546) and the K-BNT . The responders to depression treatment in the LLD group showed a significant improvement in the MMSE-KC  and Go/No-Go test . In contrast, in non-responders to depression treatment of the two groups (AD + D and LLD), there was no difference in pre- and post-treatment scores in any cognitive domains.Table t = 4.911, p = 0.000, Cohen’s d = 1.29) and Suicidality  compared to AD − D. The responders to depression treatment in AD + D showed a significant decrease in scores on the NPI , SIS , and Suicidality   and an improvement in scores on the WHO-5   showed greater deficit in some domains, such as language function in AD or executive function in LLD, than those without depression  Table . AD + D Previous studies found that depression impacts the cognitive progression of AD  and thatAt baseline, in our study, the AD + D group had more impaired language function than the AD − D group, but it was unclear whether language dysfunction was caused by neurodegenerative changes or temporarily damaged due to depression. After depression treatment, a significant improvement in language function was observed in AD + D compared to AD − D; hence, it is plausible that some degree of language impairment in AD + D stems from depression. This finding implies that language function in AD might be vulnerable to depression, which could contribute to poor prognosis of AD. Despite the high prevalence of depression in AD, the evaluation and treatment of depression in clinical settings is often overlooked. Our findings suggest the importance of evaluation and treatment of depression in AD patients, since comorbid depression in AD could contribute to a decline in cognitive functioning.Cognitive domain influenced by depression in AD + D was different from those with LLD. It is presumed that regions of brain affected by depression may differ between AD and LLD. In AD, the medial temporal lobe of the brain is the first area to exhibit atrophy , 27, 30,Also, in LLD, we identified depression may impact the executive function. There are previous reports of frontal lobe abnormalities in LLD , 54. In AD + D had lower response rates to depression treatment than LLD  Table , which iOverall, this study demonstrated that the patterns of depression seen in AD differed from typical depression seen in the elderly without dementia, specifically regarding the affected cognitive domains and antidepressant response rate. From our results, depression in AD might be thought of as a different subtype of depression. Future studies should explore the possibility of differences in pathophysiology between AD + D and LLD to determine whether depression in AD patients requires a different therapeutic approach. Moreover, this study demonstrated that depression treatment had beneficial effects on decreasing behavioral disturbances and suicide-related factors in AD patients with depression, and improving ADL and QOL in the elderly with LLD Tables , 4.This study had several strengths. We identified language impairment as a specific cognitive domain impacted by depression in those with AD, which improved through the treatment of depression via SSRIs. This is the first study to examine the specific cognitive domains impacted by AD and comorbid depression. We also found that a different cognitive function, specifically executive function, are affected in patients with late-life depression, but without dementia. Our research thus suggests that different therapeutic approaches may be required when treating depression in AD patients, compared to those required for treating patients without dementia. Our study, could consider the low antidepressant response rates in the AD patients with depression compared to those in patients with late-life depression, to support the concept that the pathogenic mechanism of AD-related depression could be different from depression in the elderly with normal cognition.Our study had a few limitations. The small sample size might have limited the generalizability of the results. In the future, studies with larger samples are needed to determine the effect of depression treatment on cognitive outcomes in AD patients. Another limitation was that the healthy control group had a significantly higher level of education than that of the LLD group. Although we statistically adjusted for age, sex, and education when analyzing cognitive function between the groups, considerable differences in the level of education might actually have influenced the psychometric assessment.Nevertheless, the current study has demonstrated the effectiveness of treatment for depression in AD patients as well as elderly population without dementia regarding improvement in cognitive functioning (language and executive functioning). This is valuable as a frontier study in examining of the association between depression treatment and cognitive outcomes in AD.In conclusion, our study demonstrated that depression treatment positively influences not only cognitive function but also overall functioning and other psychosocial factors in AD and LLD. Our findings have important clinical implication for diagnosis and treatment of depression in both AD patients and the elderly population without dementia.


Math anxiety severely impacts individuals’ learning and future success. However, limited is understood about the profile in East Asian cultures where students genuinely show high‐level math anxiety, despite that they outperform their Western counterparts. Here, we investigate the relation between math anxiety and math achievement in children as young as first and second graders in Taiwan. Further, we evaluate whether intensive exposure to digital game‐based learning in mathematics could ameliorate math anxiety.The study first evaluated a group of 159 first and second graders’ math anxiety and its correlation with math performance. Subsequently, a quasi‐experimental design was adopted: 77 of the children continued and participated in multi‐component digital game training targeting enumeration, speeded calculation, and working memory. Post‐assessment was administered afterward for further evaluation of training‐associated effects.Results confirmed that math anxiety was negatively associated with school math achievement, which assessed numerical knowledge and arithmetic calculation. Furthermore, children's math anxiety was remarkably reduced via digital training in mathematics after 6‐week intensive remediation. Crucially, this math anxiety relief was more prominent in those with high‐level math anxiety. Although the children who underwent the training showed training‐induced math achievement and working memory enhancement, this cognitive improvement appeared to be independent of the math anxiety relief.Our findings demonstrate that students can show highly negative emotions and perceptions toward learning even in high‐achieving countries. Auspiciously, the feeling of distress toward learning has the feasibility to be relieved from short‐term intensive training. Our study suggests a new approach of early treatments to emotional disturbance that can lead to permanent consequences in individuals. First and second graders show negative association between math anxiety and math performance. After 6‐week intensive remediation, children with high initial math anxiety showed remarkable reduce in math anxiety level. Furthermore, the worse their math anxiety is, the greater these children benefit from math anxiety remediation. In Taiwan, formal math curriculums are not introduced until the elementary school stage. Thus, our participants were in the starting stage of mathematical learning. To avoid data being nested and to include children from families with a wide range of socioeconomic status, we recruited participants individually from multiple school districts in Taipei, Taiwan using mailing to schools and posting at community groups rather than examining all students within one classroom. The sample size in this study was beyond the desired number of 148, as derived from a prospective power‐calculation using the software G*Power to reach the power of 1 − β = .95 at α = .05 based on the effect size (r = −.29) found in the study of Gunderson et al. , with an age range from 6.41 to 8.71 , indicating high internal consistency of the math anxiety measurement. Cronbach's alpha administered on the 77 children being analyzed for training effects was .75  at the initial assessment and .83  at the post‐training assessment. These assessments showed that the internal consistency was stable over time.To assess children's math anxiety levels, we adapted Child Math Anxiety Questionnaire (CMAQ) , which can be administered to participants aged between 6 and 16  = 3.18, p = .204). There were significant group differences in math anxiety  = 5.45, p = .005, ηp2 = .07), arithmetic calculation  = 22.99, p < .001, ηp2 = .23), and a marginal effect in numerical knowledge  = 2.98, p = .054, ηp2 = .04). Post hoc comparisons using Tukey's HSD suggested that group differences in math anxiety were primarily elicited from children of the middle of Grade 2 showing a higher level of math anxiety than children assessed at the end of Grade 2 (p = .007). Group differences in calculation were primarily elicited from the enhancement between children from Grade 1 and those from the middle of Grade 2 (p < .001). No other group differences were observed. Because of the tight age range and the limited sample size, participants from the three groups were pooled for subsequent analyses.The descriptive demographic data and cognitive assessments of the children comprising age, math anxiety, scaled IQ measures, math skills , and verbal working memory are presented in Table 3.2r(157) = −.45, p < .001, 95% CI ). After controlling for age and the IQ measures using hierarchical regression analysis, the relation between math anxiety and math achievement remained significant . We further examined the relations in each of the math achievement subtests. Negative relations with math anxiety were observed in both numerical knowledge (r(157) = −.43, p < .001, 95% CI ) and arithmetic calculation (r(157) = −.40, p < .001, 95% CI ) subtests. While controlling for age and the IQ measures, the negative correlations remained significant .We examined the relationship between math anxiety and math achievement. Zero‐order correlation between the initial math anxiety and math achievement was conducted and is presented in Figure 3.3χ2(1) = 1.88, p = .170; low‐intensity: Kruskal–Wallis χ2(1) = 2.06, p = .151).To raise ecological validity, children who participated in the training session were encouraged to play the training game at home after school 30 min a day, at least 3 days a week for 4 to 6 weeks. Children who maintained compliance and participated in the full training session for more than 6 h in total were included as the high‐intensity training group. Children who were exposed to the game for less than 2 h with a matched interval between the initial and post‐training assessment were included as the low‐intensity training group. These criteria resulted in a sample of 40 children in the high‐intensity group and 37 children in the low‐intensity group. Demographic data of the two groups were examined and are presented in Table nparLD. Two‐way interaction between time and training intensity was detected , whereas math anxiety slightly increased in the low‐intensity group . After training, the high‐intensity group showed lower math anxiety than the low‐intensity group  = 14.80, p < .001). Considering the variation of individuals at the initial assessment, we conducted an ANCOVA to compare post‐assessment math anxiety between the two groups adjusted by both initial math anxiety and math performance. Results indicated the training‐related math anxiety relief persisted after controlling for mathematical anxiety and performance at baseline  = 22.20, p < .001, ηp2 = .23).Next, we examined the remediation effect in math anxiety elicited by the computer‐based training program. Since the normality assumption was not met in the data, we conducted nonparametric analyses of the factorial design using ANOVA‐type statistics (ATS)  and a marginally significant interaction on the school setting‐related math anxiety subscale . To examine whether sex bias existed in the training‐associated relief of math anxiety, a three‐way ANOVA‐type nonparametric statistic for training intensity, time, and sex was conducted. Results indicated the math anxiety relief did not differ between male and female participants .We further examined whether different types of math anxiety (problem solving‐related and school setting‐related math anxiety) showed differential remediation effects by conducting a parallel analysis on each math anxiety subscale. The results revealed a significant two‐way interaction effect on the problem‐solving related math anxiety subscale  but not the low anxiety group . Note that the training durations of the high and low math anxiety groups were not different  = 2.46, p = .117), suggesting that the math anxiety alleviation did not result from their unequal engagement in training. To ensure such an interaction was not due to the regression‐to‐the‐mean effect, we performed the same analyses on the low‐intensity training group. The median‐split approach resulted in 19 high and 18 low math anxiety participants. Importantly, the group‐by‐time interaction pattern of the low‐intensity training group was different from the high‐intensity training group , whereas those with low math anxiety showed increased math anxiety , implying the math anxiety remediation was contributed by the digital training rather than confounded by the regression‐to‐the‐mean.To further investigate whether children who suffered from different levels of math anxiety showed differential math anxiety remediation effects, we divided children into high and low math anxiety groups based on the initial math anxiety of the high‐intensity group using a median split. This procedure resulted in 20 high and 20 low math anxiety children in the high‐intensity training group. Results revealed a marginally significant interaction between the initial math anxiety level (high vs. low) and time  = −.51, p < .001, 95% CI ). A parallel analysis was conducted on children from the low‐intensity training group to confirm the specificity of such a relation. Results indicated the initial math anxiety was not significantly associated with the changes of math anxiety in the children who minimally attended the training, if at all (r(35) = −.30, p = .070, 95% CI ; Figure We then examined whether there was an individual difference in math anxiety relief. Math anxiety relief was calculated by first subtracting initial math anxiety from post‐training math anxiety and then divided the given value by the standard deviation of initial math anxiety. We assessed the correlation between math anxiety relief and initial math anxiety for children from the high‐intensity training group Figure . Critica3.5F = 3.47, df = 1, p = .063; Table F = 4.83, df = 1, p = .028), whereas the low‐intensity group did not show the effect . Yet, the post‐training working memory between the high‐ and low‐intensity training groups was not statistically different  = 1.48, p = .223).We then examined whether verbal working memory capacity was improved by the 6‐week digital training on working memory performance. The interaction between group and time was marginally significant , whereas the low‐intensity group showed no difference in math achievement between the initial and post‐training assessment . The post‐training difference in overall math achievement between these two groups, however, did not reach significance  = 1.75, p = .186). We further examined whether the two math achievement subtests  showed differential training effects. The results revealed that for the numerical knowledge subtest, there was no training intensity‐by‐time interaction  but not the low‐intensity group . No difference was detected between these two groups at post‐assessment in either the numerical knowledge subtest  = 2.30, p = .130) or the calculation subtest  = 0.58, p = .447).Parallel analyses were conducted on children's math achievement. Similarly, the interaction effect between group and time was significant (ATS r(38) = .26, p = .103, 95% CI ; numerical knowledge: r(38) = .03, p = .840, 95% CI ; arithmetic calculation: r(38) = .02, p = .909, 95% CI ). Further, we did not find significant correlation between the training‐associated improvement in math achievement and the math anxiety relief  = −.28, p = .082, 95% CI ; arithmetic calculation: r(38) = −.16, p = .320, 95% CI ). The association between working memory improvement and math anxiety relief was not significant either (r(37) = −.20, p = .220, 95% CI ) after excluding one outlier (beyond 2.5 standard deviations from the means for both measures). These results suggested that the improvement of cognitive abilities could neither predict the initial math anxiety nor the training‐associated math anxiety relief.Finally, we examined whether individual differences of the performance gain were associated with the math anxiety profiles. Results indicated the training‐associated improvement in working memory, numerical knowledge, and arithmetic calculation was independent of the initial math anxiety level  have reported learning motivation does not predict math anxiety in first and second graders in the first three months of the school year. We, therefore, suspect that differences in the motivation of participants are plausibly negligible. Another problem introduced by the quasi‐experimental design could be the regression‐to‐the‐mean effect due to the lack of a control group. Yet, we have demonstrated that math‐anxious children have shown math anxiety relief only when they are highly engaged in our digital training, whereas those with minimal engagement remained math‐anxious. These findings suggest the remediation effect on math anxiety in the current study should likely be a real effect rather than a regression artifact.Another limitation is it would be arguably better to include a control group with a different kind of training. An active control group can help eliminate placebo effects and locate the sources of the remediation effect by elucidating whether it results from prolonged exposure to math materials or the motivating game‐based environment. Nevertheless, Szűcs and Myers  argued t5In conclusion, this study speaks to parents and educators to pay attention to students’ perceptions, emotions, and attitudes toward learning, even in the cultures where students are generally competent in mathematics. Negative attitudes toward learning can cause disruptions to students’ learning and further discourage students from being well performed (Rattan et al., The authors declare no conflicts of interest.https://publons.com/publon/10.1002/brb3.2557The peer review history for this article is available at SUPPORTING INFORMATIONClick here for additional data file.


In Diamond‐Blackfan anaemia (DBA), iron overload (IO) is common in transfusion‐dependent patients, yet has also been reported in non‐transfusion‐dependent patients. We explored the incidence of IO in transfusion‐dependent and non‐transfusion‐dependent DBA patients. We observed hepatic IO in 65% of patients analysed with MRI, including three patients that were only treated with transfusions in the past. Whereas overall ferritin levels and liver iron content correlated, ferritin levels did not reflect total body iron adequately. Our data suggest that transfusion burden in the past plays an important role in IO in DBA, and should be taken into account during follow up. Treatmeo cancer . Secondlo cancer , 4, 5. To cancer . Howevero cancer , 8. Nowao cancer , 9, 10.In DBA, RBC transfusions are the main cause of IO . StudiesHere we report the incidence and severity of IO in a well‐described cohort of transfusion‐dependent and non‐transfusion‐dependent DBA patients with the aim to improve the diagnostic evaluation and overall management of IO in DBA.In this retrospective, observational multicentre study, we have included paediatric and adult DBA patients of whom serum ferritin level and/or MRI results were available. Patients were classified as transfusion‐dependent if they had received 10 or more RBC transfusions during the last 12 months prior to iron evaluation.Twenty‐nine patients were included, including seventeen patients 17/29, 59%) in which MRI analysis of IO had been performed. The median age was 12 years (range 1–47 years), nine patients  were male and in twenty‐four  of the patients a molecular defect had been confirmed. Based on our criteria, ten patients  were transfusion‐dependent and nineteen  were non‐transfusion‐dependent. MRI results were available in nine of the transfusion‐dependent patients  and in eight of the non‐transfusion‐dependent patients . In the other patients, MRI analysis was not performed based on either clinical, biochemical or psychological reasons, assessed by the treating physician. Patient characteristics are summarized in Table 7/29, 59%Hepatic IO was present in eleven  patients that were analysed by MRI. In the majority, , a LIC >7 mg/g indicated moderate to severe IO. Figure p < 0.0001) and mean LIC value .  were diagnosed with moderate to severe hepatic IO, all were treated with chelation therapy. Interestingly, in the non‐transfusion‐dependent group, hepatic IO was present in three  patients  and none of these patients received chelation therapy. In this group, in one patient (patient 17), cardiac T2* was 20 ms, suggesting borderline cardiac IO. However, it should be noted that the image quality in this patient was suboptimal due to motion artefacts. As expected, comparing iron parameters between transfusion‐dependent and non‐transfusion‐dependent patients revealed a significant difference in both mean serum ferritin level , all patients had been treated with RBC transfusions during the course of disease: five patients  had received less than 10 RBC transfusions, and twelve patients had received more than 10 RBC transfusions during their lives . Within this last group, eleven  patients were diagnosed with hepatic IO, in contrast to none in the group of patients who had received less than 10 transfusions. The non‐transfusion‐dependent patients with hepatic IO  were the only patients that received more than 10 RBC transfusions in the group of non‐transfusion‐dependent patients that underwent MRI analysis . Mean serum ferritin levels and mean LIC values were significantly higher in patients with a high transfusion burden (443 ng/ml vs. 57 ng/ml (p < 0.0001) and 10.29 mg/g versus 0.96 mg/g (p < 0.001), respectively) , 1–10 transfusions , and ≥10 transfusions . Of the patients evaluated with MRI  Figure .r = 0.825, p < 0.001). Interestingly, while all transfusion‐dependent patients were treated with iron chelation therapy, and serum ferritin levels did not exceed 1000 ng/ml  patient registry. Written informed consent was obtained from all patients and/or legal guardians.Figure S1. Serum ferritin and liver iron content in patients with Diamond‐Blackfan anaemia.Click here for additional data file.


Glycine max L. Merril), the effects on the soil fungal community when two legumes are involved in the crop system remain unclear. Thus, the effects of the cover crop on the soil fungal community under a succession of soya and a succession of maize (Zea mays L.) were clarified: a continuous wheat (Triticum aestivum L.)–maize cropping system was converted to new rotation systems with three cover crop treatments: leguminous vetch (Vicia sativa L.), a mixture of vetch and rye , and fallow, succeeded by soya or maize in this study. The soil fungal community at the harvest of soya and maize were determined using high-throughput sequencing of ITS2 amplicons. Compared to a wheat–maize rotation system, all of the new rotation systems that involved leguminous crops or fallow increased the soil fungal diversity and suppressed pathotrophs by reducing the soil NH4+, NO3−, available K, and available P concentrations. Different cover crops changed the fungal community composition, but their effect was overwhelmed by the strong effect of succeeding soya, which induced minor shifts among the cover crop treatments under soya than maize. The Vetch–Soya system exhibited the highest fungal diversity, which have been due to an increase of symbiotrophs. Replacing wheat with mixed vetch and rye most greatly suppressed the pathotrophs, and this suppression effect was stronger when succeeded by maize than by soya. These results showed the short-term benefits of legume–legume succession and legume–cereal mixed cover crops for increasing fungal diversity and suppressing pathotrophs. Further study is needed to examine the long-term effects of Vetch–Soya on the accumulation of legume-associated pathogens.The enrichment of soil-borne fungal pathogens and a high input of mineral fertilizer in the continuous cropping of cereal crops have raised a concern about soil health deterioration. Conversion of continuous cereal cropping to a legume-involved system alters the soil fungal community. However, when a leguminous cover crop is grown with a succeeding legume grain crop such as soya ( Gaeumannomyces graminis var. tritici, Fusarium graminearum, and Fusarium moniliforme, as well as symbiotrophs such as Arbuscular mycorrhiza . As a cover crop, a legume can improve the yield of the succeeding cereal crop and soil health by reducing soil erosion, increasing soil water and nutrient availability, and controlling soil-borne pathogens (Glycine max L. Merril) rotated with a cereal crop can maintain sustainable crop production and significantly increase the soil microbial biomass  mixed with the cereal crop rye  as cover crops, succeeding soya and maize as grain crops. The objectives of this study were (1) to determine the short-term effects of converting the prior wheat–maize cropping system to the new legume-involved crop rotation systems on the soil fungal diversity and community composition, and (2) to compare the effects of the cover crop on the soil fungal community under a succession of maize and soya. It was hypothesized that (1) the soil fungal diversity and community composition in the systems with leguminous cover crops would differ from that of wheat–maize system; (2) a more diverse cropping system would recruit more saprotrophs and symbiotrophs and suppress pathotrophs, and the cover crop effect would be different under succeeding maize than under succeeding soya.Accordingly, a field experiment was undertaken to convert a prior wheat–maize rotation system to a new cropping system involving leguminous crops or winter fallow, which included hairy vetch . For the four decades prior to the experiment, the cropping system was wheat followed by maize and regular chemical fertilizers were applied. Winter wheat was grown from the middle of October to early June, with 255 kg N ha−1, 112.5 kg P2O5 ha−1, and 75 kg K2O ha−1 applied. Summer maize was grown from early June to early October, with 180 kg N ha−1, 90 kg P2O5 ha−1 and 120 kg K2O ha−1 applied.The field experiment was established in October 2017 at the experimental station of the Farmland Irrigation Research Institute, Chinese Academy of Agricultural Sciences, Xinxiang City of Henan Province  in China. The area has a typical warm temperate continental monsoon climate, with a mean annual precipitation of 573.4 mm and a mean annual air temperature of 14.0°C. The soil was a sandy loam , a mixture of vetch with cereal rye , and fallow without a cover crop , following two summer grain crops (Maize and Soya). In total, six treatments were employed in this study, designated as Fallow–Soya, Vetch–Soya, VetchRye–Soya, Fallow–Maize, Vetch–Maize and VetchRye–Maize.The field experiment was laid out in a completely randomized design with four replicates, and the plots were 8 × 6 m . The cro−1 for Vetch, and 60 kg vetch ha−1 and 30 kg rye ha−1 for VetchRye. The above-ground biomass of winter cover crops in each plot was sampled at three randomly selected sites in an area of 0.25 m2 prior to chopping and incorporating them into the 10–15 cm soil layer using a rotary on 25th April 2018.Winter cover crops were sown on 15th October 2017 at a seeding rate of 90 kg ha−1 (40 cm between rows and 11 cm between plants in rows) or 60,000 maize plants ha−1 (60 cm between rows and 25 cm between plants in rows). No fertilizers and pesticides were applied to either winter or summer crops. Weeds were controlled manually in all plots. Soya plants suffered from the ever-green disorder  and maize (cv. Zhengdan 958) were sown at 15200 soya plants hadisorder , and no disorder . After dSoil samples were taken prior to the experiment from the 0 to 20 cm layer using an auger with an inner diameter of 3.5 cm from nine randomly selected sites on 10th October 2017 to make three composite soil samples. Soil samples after the experiment were taken using the same method from two sites between plants in each plot at the harvest of soya and maize on 3rd October 2018, resulting in 48 soil samples for each rotation system (3 cover crops × 2 grain crops × 4 replicates × 2 samples per plot).The soil samples were stored in polyethylene bags with ice packs (4°C) and transferred to the laboratory. After removing the fine litter, root residue, and stones, part of the samples was ground to pass through a 2 mm sterilized sieve and stored at −20°C for DNA extraction. The others were used to measure the soil water content, soil pH and the content of organic carbon (C), nitrogen (N), phosphorous (P), and potassium (K). The soil water content was measured using an oven-drying method. Soil pH (soil to water 1: 2.5) was measured using a pH meter (Sartorius PB-10). Soil organic C and total N were measured using an elemental analyzer  after removing the inorganic C using a 1 M hydrochloric acid solution. Ammonia and nitrate were measured using a continuous flow autoanalyzer , 15.5 μl ddH2O, and 1 μl template DNA (20 ng μl−1), followed by 10 cycles at 98°C for 30 s, 50°C for 30 s, and 72°C for 1 min, and then 25 cycles at 98°C for 30 s, 52°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 5 min. After amplification, the final DNA concentration and purification were determined using a Qubit 3 Fluorometer (Invitrogen). The DNA extraction quality was checked using 1% agarose gel electrophoresis. The purified PCR products were mixed at an equimolar ratio for sequencing using DNBSEQ-G400  following the manufacturer’s instructions. The internal transcribed spacer 2 (ITS2) region of the fungal genes was amplified with the forward primer ITS7 (5′-GTGARTCATCRARTYTTTG-3′) and the reverse primer ITS4 (5′-CCTSCSCTTANTDATATGC-3′) with a 6 bp barcode at the 5′ end for sample splitting . PolymerSEQ-G400  with theSnakemake pipeline , which was performed using the adonis function of the vegan package  was performed using the randomForest function of the randomForest package to identify which environmental factors were the main drivers of the Richness index, Shannon index, and main functional guilds , with relatively high levels of soil available nutrients (3− contents of new rotation systems were significantly decreased (p ≤ 0.001), while the soil N/P, K/P, and NO3−/K were significantly increased (p ≤ 0.001).In the wheat–maize rotation system prior to the experiment, the aboveground biomass of wheat was significantly higher than that of Vetch and VetchRye (utrients . After t−1), which was 34.2 ~ 49.2% higher than Vetch. Compared to the Fallow treatment, VetchRye significantly increased the plant biomass of succeeding Soya but not of Maize, while Vetch had no effect on the plant biomass of Soya or Maize. Soil water, the available K, and NO3− contents, K/P, and NO3−/K were significantly affected by the cover crop, grain crop, and their interaction (p ≤ 0.05). Available P was affected by the cover crop and the grain crop (p ≤ 0.001), while soil NH4+ and pH were only affected by the cover crop and the grain crop (p ≤ 0.05), respectively. With the same cover crop, soil pH, water, and available P contents were lower under Soya than Maize. On the contrary, soil available K and NO3− contents were higher under Soya than Maize, when the winter treatments were Vetch and Fallow. Soil available P and NH4+ contents were the highest in Fallow and the lowest in VetchRye followed by both Maize and Soya. The soil water and available K contents were the highest in VetchRye, and the soil water, available K and NO3− contents were the lowest in Fallow followed by Maize. The soil NO3− content was the lowest in VetchRye followed by Soya.Among the new rotation systems, VetchRye produced the highest above-ground biomass (2949.5 ~ 3044.4 kg hap ≤ 0.05) increased fungal Shannon index to an average of 5.3  than Soya , irrespective of system prior to and after the experiments  showed different correlation patterns from that of the most abundant pathogen Fusarium, but showed similar significant correlations with specific environmental factors to that of some saprotrophs and symbiotrophs , such as negative correlations with cover crop biomass and NO3−, and positive correlations with K/P and NO3−/K. The saprotrophic Mortierella was positively correlated with grain crop biomass, soil water content, and NH4+, while Actinomucor was positively correlated with SOC, TN, and the N/P and K/P ratios.To identify the environmental factors  influencing fungal diversity and community composition, the primary controlling environmental factors were identified . The bio/N ratio . For theiotrophs . The enviotrophs , defined3−, and NH4+ concentrations than the prior system, mainly attributed to the cessation of mineral fertilization in new rotation systems. This is also supported by Consistent with our first hypothesis that if the prior wheat-maize system was changed to a new rotation system involving legumes, then soil fungal community would change along with the plant biomass and soil physicochemical properties, both the soil physicochemical properties and the fungal community were strongly affected by the change of the rotation system ; Table 13− were identified as the primary factors affecting fungal diversity  can be found at: SYu: formal analysis, data curation, writing–original draft, and visualization. TW: validation, writing–review and editing, and investigation. YM: writing–review and editing. SYa: conceptualization, project administration, and funding acquisition. LW: validation and investigation. HZ: formal analysis and data curation. YZ: resources. ZS: conceptualization, methodology, software, and validation. BZ: conceptualization, methodology, writing–review and editing, and project administration. All authors contributed to the article and approved the submitted version.This study was financially supported by the National Natural Science Foundation of China (No. 4207071613), National Key Research and Development Program of China (2021YFD1700200), and China Agriculture Research System (No. CARS-04).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Rearrangement of the actin cytoskeleton is critical for cytotoxic and immunoregulatory functions as well as migration of natural killer (NK) cells. However, dynamic reorganization of actin is a complex process, which remains largely unknown. Here, we investigated the role of the protein Cereblon (CRBN), an E3 ubiquitin ligase complex co‐receptor and the primary target of the immunomodulatory drugs, in NK cells. We observed that CRBN partially colocalizes with F‐actin in chemokine‐treated NK cells and is recruited to the immunological synapse, thus suggesting a role for this protein in cytoskeleton reorganization. Accordingly, silencing of CRBN in NK cells results in a reduced cytotoxicity that correlates with a defect in conjugate and lytic synapse formation. Moreover, CRBN depletion significantly impairs the ability of NK cells to migrate and reduces the enhancing effect of lenalidomide on NK cell migration. Finally, we provided evidence that CRBN is required for activation of the small GTPase Rac1, a critical mediator of cytoskeleton dynamics. Indeed, in CRBN‐depleted NK cells, chemokine‐mediated or target cell–mediated Rac1 activation is significantly reduced. Altogether our data identify a critical role for CRBN in regulating NK cell functions and suggest that this protein may mediate the stimulatory effect of lenalidomide on NK cells. Cereblon (CRBN), the primary target of the Immunomodulatory drugs (IMiDs), regulates the activation of the small GTPase protein Rac1 and is required for NK cell migration and cytotoxicity. Our study reveals new information on complex mechanisms underlying cytoskeleton reorganization and stimulatory effects of IMiDs in NK cells. Natural Killer (NK) cells are innate lymphoid cells that play an important role in immune response against infection diseases and cancer, via contact‐dependent cellular cytotoxicity and cytokine and chemokine production , 2.NK cell cytotoxicity is a multistep and tightly controlled process. Following NK cell activating receptor engagement, an early step is NK cell conjugation with the target cell followed by formation of the immunological synapse (IS) at the contact site, lytic granule polarization, and degranulation. Cell adhesion molecules, in particular the integrin lymphocyte function‐associated antigen‐1 (LFA‐1), play an essential role in these mechanisms. The engagement of LFA‐1 with its ligand ICAM‐1 mediates the firm adhesion to the target cell and drives the accumulation of filamentous actin (F‐actin) as well as lytic granule convergence toward the microtubule organizing center (MTOC) and polarization at IS , 4. MoreAdhesion molecules, chemokines, and cytoskeleton rearrangement are also important regulators of NK cell migration. In particular, the binding of chemokines to their receptors triggers a complex signaling cascade that supports leukocyte migration, regulates the integrin adhesiveness, and causes a fine remodeling of cytoskeleton components. Key regulators of NK cell cytotoxicity and migration are the RhoA, Rac1, and Cdc42 small GTPases. In migrating cells, Cdc42 and Rac1 cause actin polymerization leading to the formation of filopodia and lamellipodia, respectively , 8. MoreSeveral studies described the capability of immunomodulatory drugs  to regulate NK cell function by stimulating the expression of different activating receptors , cytokinCRBN) complex, containing also cullin‐4 (CUL4), the DNA damage binding protein‐1 (DDB1), and the RING‐finger protein (ROC1). Moreover, CRBN can function in a ubiquitin‐independent manner by binding and modulating the membrane expression and/or the function of different proteins [CRBN complex [A key target of these drugs is the protein cereblon (CRBN) , even ifproteins . Through complex , 21 or d complex . IMiDs c complex . Indeed, complex .+ T cell activation via epigenetic repression of the Kv1.3 potassium channel expression required for calcium influx [+ T‐cell effector responses through c‐myc‐dependent regulation of the central metabolism [In the context of immune cells, CRBN was shown to limit murine CD4m influx , and to m influx . Moreovetabolism . A studytabolism .In this study, we show that CRBN is required for NK cell migration and cytotoxicity. We demonstrate for the first time that CRBN partially localizes with accumulated F‐actin in chemokine‐treated NK cells or at IS and can regulate Rac1 activation. Taken together, these observations indicate a role for CRBN in the regulation of key NK cell functions and suggest that lenalidomide‐mediated alteration of CRBN activity is responsible for its stimulatory effect on NK cells.Little evidence has been provided so far on the role of protein CRBN in NK cells . By westOn the basis of these observations, we examined whether a different stimulus known to induce cytoskeleton reorganization, such as a chemokine, could affect CRBN cellular distribution. We observed that CRBN localized to the lamellipodial leading edge and partially overlapped with F‐actin in CXCL12‐treated NK cells Fig. .These findings indicate that stimuli known to induce cytoskeleton remodeling can also cause a cellular redistribution of CRBN in NK cells.51Cr‐release assay. We observed that loss of CRBN expression resulted in a significant reduction in NK cell cytotoxicity relative to the scramble control .The human K562, NK‐92, and HEK293T cell lines were purchased from ATTC and were maintained at 37°C and 5% COHealthy donors’ peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Nycomed) gradient centrifugation. NK cells were negatively selected from PBMCs using MACS human NK cell isolation kit (Miltenyi Biotec).Lenalidomide was purchased from BioVision, Inc. , and NSC23766 and MLN4924 were purchased from Merck Life Science. Homo‐PROTAC cereblon degrader 1 was purchased from Selleckchem. The following monoclonal antibodies (mAbs) were used for immunostaining: anti‐CD3/FITC, anti‐CD56/PE, anti‐CXCR4/APC, anti‐GZM‐B/FITC and anti‐GZM‐B/APC, anti‐total LFA1/APC (BD Biosciences), anti‐LFA1 clone 24 CD11‐CD18 (Hycult Biotech), and APC Goat anti‐mouse IgG (Jackson Immunoresearch Laboratories). Phalloidin‐FITC was purchased from Thermo Fisher Scientific .Human recombinant cytokines IL‐2 and CXCL12 were purchased from Peprotech EC.Total RNA was extracted using RNA isolation Kit (Norgene) and used for cDNA first‐strand synthesis in a 25 μL reaction volume according to the manufacturer's protocol for M‐MLV reverse transcriptase (Promega). cDNAs were amplified in triplicate with primers for CRBN (Hs00372271_m1), GZM‐B (Hs00188051_m1), ROC1 (Hs00360274_m1), and GAPDH (Hs03929097_g1) conjugated with fluorochrome FAM (Applied Biosystems) using the ABI Prism 7900 Sequence Detection system (Applied Biosystems). The level of expression was measured using Ct (threshold cycle) as previously described .For knocking down CRBN in NK‐92 cell line, we used a pLKO.1‐shRNA‐CRBN (TRCN0000141562) lentiviral vector with puromycin resistance and the control vector pLKO.1 non‐targeting shRNA (MISSION Merck Life Science). For knocking down CRBN expression in primary NK cells, we used pLKO.1‐shRNA‐CRBN‐GFP‐lentivirus vector and the control vector pLKO.1 non‐targeting shRNA GFP‐lentivirus vector gently provided from Dr K.A. Stewart . For kno6 NK cells in 200 μL complete medium with Polybrene (8 μg/mL) (Merck Life Science) for 2 h. For GFP‐expressing viruses, the efficiency of infection was evaluated after 3 days by measuring GFP expression using FACS analysis. After infection, NK‐92 cells were allowed to expand for 24 h and were then selected for puromycin resistance (2 μg/mL). In some experiments, NK cells infected were sorted for GFP expression at day 3 postinfection using FACSAria III (BD Bioscience) equipped with a 488 nm laser and analyzed using a FACSDiva Software (BD Bioscience version 6.1.3).For lentivirus production, 5 μg of viral DNA was transfected into semi‐confluent HEK293T cells using Lipofectamine Plus (Thermo Fisher Scientific) as previously described . After 7β‐actin and CRBN were purchased from Merck Life Science. Antibody against lamin A, tubulin, IKZF1, and IKZF3 were purchased from Santa Cruz Biotechnology. An HRP‐conjugated secondary Ab and an ECL detection system  were used to reveal immunoreactivity following the manufacturer's instructions.For western blot analysis, total and nuclear proteins from primary NK cells were prepared as previously described . AntibodRac1 activation assay was performed as previously described .Purified NK cells from healthy donors or NK cells infected with lentivirus pLKO.1‐shRNA‐CRBN‐GFP or nontarget shRNA were treated for 18 h with 1 μM lenalidomide or DMSO in the presence of IL‐2 (200 U/mL) in complete medium RPMI 1640 10% FBS. Cell migration and chemokine‐induced expression of β2 integrin activation‐neoepitope was assayed as previously described . The perThe surface expression of CXCR4 was analyzed on freshly isolated NK cells after treatment of 18 h with 1 μM lenalidomide or DMSO in the presence of IL‐2 (200 U/ml) or on infected cells (primary NK cells and NK‐92 cell line) by immunofluorescence staining using anti‐CXCR4/APC and mouse IgG1/APC isotype control Ab. Fluorescence was analyzed using a FACSCanto II flow cytometer.Scramble and CRBN silenced primary NK or NK‐92 cells were mixed with Cell Tracker Deep Red‐loaded K562 target cells at a 1:2 ratio. Cells were pelleted at 1000 g for 1 min and incubated at 37°C for different times. The cells were slowly resuspended five times and immediately fixed with 4% PFA for 30 min at room temperature. Then, cells were washed with PBS and stained with anti‐CD56/PE mAb for 20 min. Flow cytometry was used to measure conjugate frequency. Conjugates were identified as double positive cells (PE/Deep Red) on total or gated GFP positive cells for NK‐92 and primary NK cells, respectively.A standard 4‐h chromium‐release assay using as effector cells pLKO.1‐scramble or pLKO.1‐CRBN shRNA NK‐92 cells was performed as previously described .l‐lysine‐coated multichamber slides in the presence of CXCL12 for 30 min at 37°C. For analysis of CRBN at NK cell IS, primary NK cells or NK‐92 cells were incubated with K562 target cells in a 1:1 ratio for 7 min a 37°C. Cells were resuspended, allowed to adhere and fixed as previously described [For chemokine stimulation, we allow primary NK cells to adhere on poly‐escribed , stainedt‐test. Differences between multiple groups were analyzed with two‐way analysis of variance (ANOVA). Prism 6 (GraphPad) software was used. Graphs show mean values, and error bars represent the SD.Statistical significance between two groups was determined by performing two‐tailed, paired Student's The authors declare no commercial or financial conflict of interest.Human peripheral blood was obtained from donors after written informed consent and in accordance with Research Ethics Committee guidelines at the Policlinico Umberto I Hospital. All Research Ethics Committee guidelines were in accordance with the Declaration of Helsinki principles.C.F. and H.S. designed research, performed the experiments, analyzed results, and wrote the manuscript; A.K., S.R., and C.C. performed the experiments and analyzed the results; G.P. performed cell sorter experiments; A.S., A.Z., and M.C. contributed with analytic tools and analyzed the results; R.M. and R.P. performed microscopic analysis; M.C. and A.G. critically reviewed the manuscript; and A.S. contributed to design research and write the manuscript. All authors have contributed and approved the final version of the paper.https://publons.com/publon/10.1002/eji.202149269The peer review history for this article is available at CRBNcereblonISimmunological synapseCRBNCLR4cullin‐4‐RING ubiquitin ligaseROC1RING‐finger proteinGZM‐Bgranzyme BIMiDsimmunomodulatory drugsIKZFIkaros transcription factorSupporting InformationClick here for additional data file.


The study aimed to analyze the relationship between expression levels of chemerin, oxidized low density lipoprotein (ox‐LDL), matrix metalloproteinase 9 (MMP‐9) and pregnancy associated plasma protein A (PAPP‐A) in ischemic cerebrovascular disease (ICVD) patients and the relationship between the mentioned indicators and the degree of neurological impairment.From January 2020 to February 2021, a total of 328 cases of ICVD patients were admitted to our hospital, and 240 cases of healthy people (control group) were prospectively recruited into this study. The 328 patients were divided into 2 ischemic subtypes, with 233 cases as acute cerebral infarction (ACI) and 95 cases as transient ischemic attack (TIA). Laboratory tests were compared among the groups. Spearman rank correlation was used to analyze the correlation between chemerin, ox‐LDL, MMP‐9, PAPP‐A levels and neurological deficit. Unconditional logisitic regression was used to analyze the risk factors for neurological deficits.p < 0.05, respectively), while the levels of the mentioned indicators in the TIA group were significantly higher than those in control group . The levels of the given indicators decreased successively in the severe, moderate, and mild neurological deficits population and control group, with statistical difference. Spearman rank correlation analysis showed that chemerin, ox‐LDL, MMP‐9, and PPAP‐A levels were positively correlated with the degree of neurological deficit in ICVD patients. Unconditional logistic regression analysis showed that chemerin, ox‐LDL, MMP‐9, and PPAP‐A were the independent risk factors for neurological deficit in patients with ICVD.The high density lipoprotein cholesterol (HDL‐C), fasting plasma glucose (FPG), chemerin, ox‐LDL, MMP‐9, and PPAP‐A levels in the ACI group were significantly higher than those in the TIA group and control group (LDL‐C, FPG, chemerin, ox‐LDL, MMP‐9, and PPAP‐A were highly expressed in ACI and neurological deficit patients. Chemerin, ox‐LDL, MMP‐9, and PPAP‐A may be the independent risk factors for neurological deficit in patients with ICVD. About 80% of patients with CVD die from ischemic cerebrovascular disease (ICVD)  levels and facilitates infiltration of arterial endothelial cells, which can contribute to atherosclerosis or narrowing of arterial walls  and transient ischemic attack (TIA) group (n = 95) according to the ischemic type. Also, based on National Institute of Health Stroke Scale (NIHSS) scores, 328 cases were divided into 3 subgroups, namely, mild neurological deficit , moderate neurological deficit , and severe neurological deficit .From January 2020 to February 2021, 328 ICVD patients and 240 cases of healthy people, who came to our hospital for physical examination at the same period, were prospectively enrolled in this study. The ICVD patients were divided into the acute cerebral infarction (ACI) group  The diagnosis of ICVD was established according to Chinese Guidelines for Cerebrovascular Diseases  Patients with severe liver or kidney function disorder; (2) with hemorrhagic cerebral vascular diseases; (3) with autoimmune diseases; (4) with severe infectious diseases or chronic inflammatory diseases within 1 month; (5) underwent surgery or suffered tissue trauma within 3 months; (6) took hypolipidemic and anticoagulant drugs within 2 months; (7) ICVD caused by tumor, hematologic disease, arteritis, cardiogenic, cerebrovascular malformation, or aneurysm; and (8) hemorrhage after cerebral infarction, or hemorrhagic stroke.3Baseline characteristics including age, gender, body mass index (BMI), history of smoking, drinking, hypertension, and diabetes were collected.Fasting venous blood was collected from all patients the next morning after admission, and from the people in the control group on the day of physical examination. The blood samples were centrifuged at 2500 r/min for 15 min, and the supernatant was taken for further analysis. The levels of low density lipoprotein cholesterin (LDL‐C), high density lipoprotein cholesterin (HDL‐C), triglyceride (TG), total cholesterol (TC), and fasting plasma glucose (FPG) were tested using the automatic analyzer . The levels of chemerin, ox‐LDL, MMP‐9, and PAPP‐A were analyzed by enzyme linked immunosorbent assay. All the earlier‐mentioned procedures were carried out in accordance with the reagent instructions.The levels of laboratory tests and NIHSS scores were compared among groups, and the correlations of laboratory indexes with the severity of neurological deficit were analyzed by Spearman rank correlation analysis. Unconditional logisitic regression analysis was performed to analyze the risk factors for neurological deficit.Evaluation of degree of neurological deficit Lyden, : NIHSS w3.1x¯±s. One‐way ANOVA was used for the comparison among groups. LSD method was used for pairwise comparison of data between and within groups. The measurement data of non‐normal distribution were expressed as M , and the rank‐sum test was used for comparison between the two groups. The count data were expressed in percentage (%) and compared with chi‐squared test. Correlation analysis was performed by Spearman rank correlation analysis. Unconditional logisitic regression was used to analyze the risk factors of neurological deficits. p < 0.05 was considered as statistically significant difference.All data were analyzed using statistical software SPSS 21.0. The database was built with Excel. The measurement data conforming to normal distribution were expressed as 44.1p > 0.05). Hypertension and diabetes between the ACI group and TIA group were of no significant difference (p > 0.05). The BMI and NIHSS scores in the ACI group were significantly higher than those in the TIA group and control group, and BMI and NIHSS scores in the TIA group were significantly higher than those in the control group . Baseline characteristics are shown in Table There were 233 cases of ACI patients, with 91 males and 142 females. They were aged from 42 to 69 years with an average age of 52.19 ± 10.63 years. There were 95 cases in the TIA group, with 37 males and 58 females. They were aged from 43 to 68 years with an average age of 52.08 ± 10.59 years. There were 94 males and 146 females in the control group. The age ranged from 42 to 70 years with an average age of 52.43 ± 10.51 years. The age, gender ratio, and history of smoking and drinking among the three groups were of no significant difference (4.2p > 0.05). The levels of LDL‐C, FPG, chemerin, ox‐LDL, MMP‐9, and PAPP‐A in the ACI group were significantly higher than those in the TIA group and control group. The levels of these indicators in the TIA group were higher than those in the control group . The comparisons are shown in Table There were no significant differences between HDL‐C, TG, TC levels among the three groups (4.3p > 0.05). The levels of LDL‐C, FPG, chemerin, ox LDL, MMP‐9, and PAPP‐A were significantly different between the severe, moderate, mild neurological deficit populations and the control group, with significant differences (p < 0.05). The comparisons are shown in Table There were no significant differences between HDL‐C, TG, and TC levels among the different severities of neurological deficit patients .Spearman rank correlation analysis showed that chemerin, ox‐LDL, MMP‐9, and PAPP‐A levels were positively correlated with the severity of neurological deficits in patients with ICVD  (see Table We included factors with difference in univariate analysis into multivariate logistic regression analysis. The results of multivariate logistic regression analysis showed that the risk factors for neurological deficits in ICVD patients were chemerin, ox‐LDL, MMP‐9, and PAPP‐A (OR = 4.652, 1.303, 1.808, 2.786; 95% CI = 1.202–7.487, 1.022–2.212, 1.031–2.721, 2.003–3.698; 5ICVD is very common in clinics, and is more common in middle‐aged and elderly patients. It is mainly caused by atherosclerosis on the basis of inflammation, which leads to cerebral ischemia and hypoxia and induces a series of neurological defects, with a high disability rate and mortality, of which more than 50% patients can be accompanied by neurological sequelae of varying degrees (Lu & Wang, Adipose tissue is an active endocrine organ in the body. The inflammatory mediators and cytokines secreted by adipose tissue, also known as adipokines, can promote or improve insulin resistance, lipid metabolism disorders, vascular dysfunction, chronic inflammation and participate in the occurrence and development of atherosclerosis (Demir et al., MMP‐9 is a member of the MMPs family, which is closely related to CVD. Under normal circumstances, MMP‐9 is underexpressed. However, in pathological conditions, the concentration of MMP‐9 increases, and MMP‐3, MMP‐8, and other enzymes are activated to synergistically degrade a large amount of extracellular matrix, weaken the fibrous cap structure, and contribute to intima thickening and plaque formation. Zhang  indicateWang et al.  suggeste5.1This is a single‐center trial, where the evidence strength is limited. The results of this trial need to be confirmed by multicenter, randomized, double‐blinded trials.6LDL‐C, FPG, Chemerin, ox‐LDL, MMP‐9, and PPAP‐A were highly expressed in ACI and neurological deficit patients. The higher the levels, the more serious the nerve deficit is. Chemerin, ox‐LDL, MMP‐9, and PPAP‐A may be the independent risk factors for neurological deficit in patients with ICVD.The authors declare that they have no competing interests.Jianpu Jia and Leguo Zhang contributed to the conception and design of the study; Lixuan Wang, Zhen Hong, and Liran Zhang performed the experiments, collected, and analyzed data; Jianpu Jia, Ruixue Xia and Leguo Zhang wrote the manuscript; Jianpu Jia, Leguo Zhang, and Zeyu Zhao revised the manuscript. All authors reviewed and approved the final version of the manuscript.