Faecalibacterium and Catenibacterium in ET. In conclusion, fecal SCFAs were decreased in ET and correlated with clinical severity and gut microbiota changes. Fecal propionic, butyric, isobutyric and isovaleric acid might be potential diagnostic and differential diagnostic biomarkers for ET.Diagnosis of essential tremor (ET) at an early stage can be difficult, especially when distinguishing it from healthy controls (HCs) and Parkinson’s disease (PD). Recently, stool sample analysis of gut microbiota and its metabolites provides new ways to detect novel biomarkers for neurodegenerative diseases. Short-chain fatty acids (SCFAs), as the main metabolites of gut microbiota, were reduced in the feces of PD. However, fecal SCFAs in ET have never been investigated. We aimed to investigate the fecal SCFA levels in ET, assess their relationships with clinical symptoms and gut microbiota, and identify their potential diagnostic abilities. Fecal SCFAs and gut microbiota in 37 ET, 37 de novo PD and 35 HC were measured. Constipation, autonomic dysfunction and tremor severity were evaluated by scales. ET had lower fecal propionic, butyric and isobutyric acid levels than HC. Combined propionic, butyric and isobutyric acid distinguished ET from HC with an AUC of 0.751 (95% CI: 0.634–0.867). ET had lower fecal isovaleric and isobutyric acid levels than PD. Isovaleric and isobutyric acid differentiated ET from PD with an AUC of 0.743 (95% CI: 0.629–0.857). Fecal propionic acid was negatively correlated with constipation and autonomic dysfunction. Isobutyric and isovaleric acid were negatively associated with tremor severity. Lowered fecal SCFAs were related to a decreased abundance of  The clinical profile of ET is characterized by not only motor symptoms but also some nonmotor features, including gastrointestinal disorders2. Numerous studies have been performed to investigate the pathological and physiological features of essential tremor; however, a clear pathophysiological mechanism has not been identified4. Recent studies have shown that dysfunction of the microbiota-gut–brain axis might lead to neurodegenerative diseases, and increasing evidence has indicated a potential bidirectional relationship between gut microbiota and neurodegenerative diseases6. It was notable that fecal microbiota transplantation simultaneously ameliorated the patient’s essential tremor and irritable bowel syndrome in a case report, which might suggest a close link between gut microbiota and essential tremor7. Furthermore, we identified specific gut microbiota alterations in ET patients, which strongly demonstrated an important role of gut dysbiosis in ET8.Essential tremor (ET) is a progressive, chronic, neurodegenerative disease mainly characterized by an action tremor of the upper limbs, possibly with the involvement of other parts of the body, such as the head, vocal cords and lower limbs5. The imbalanced microbiome enhances intestinal permeability and activates enteric glial cells, which contribute to α-synucleinopathy11. PD shares certain overlapping features with ET, such as a similar tremor frequency, overlapping resting tremors , and postural tremors (mainly in ET patients) in both ET and PD patients, making it challenging to distinguish one from the other at an early stage12. Thus, it is urgent for us to open a helpful window for distinguishing ET from PD. In this context, studying the specific gut dysbiosis and related metabolite changes in ET and identifying its differences from PD might provide potential biomarkers for ET diagnosis and differential diagnosis.Regarding gut dysbiosis in neurodegenerative diseases, PD is the most widely studied14. SCFAs are absorbed in colonocytes and transported to the liver via the portal venous system, and some SCFAs enter the systemic circulation. SCFAs have local effects in maintaining intestinal barrier integrity and shaping gut mucosal innate immunity15. They also have remote effects on the blood–brain barrier (BBB) by promoting tight junction proteins and crossing the BBB to activate neurons through stimulation of G protein-coupled receptors (GPCRs)16. Acetic acid, propionic acid and butyric acid are the most abundant SCFAs in the colon. Previous studies have identified reduced fecal levels of acetic acid, propionic acid and butyric acid in PD patients17. However, the fecal SCFA levels in ET patients have never been investigated.Short-chain fatty acids (SCFAs) are the main metabolites produced by gut bacterial fermentation of dietary fiber and are speculated to be pivotal in gut–brain cross talkThus, our study aimed to identify specific fecal SCFA alterations in ET patients and their differences from PD patients, to assess the relationships of fecal SCFAs with clinical symptoms and gut microbiota in ET and to identify the potential diagnostic and differential diagnostic ability of fecal SCFAs. To eliminate the confounding factor of anti-PD medication, we selected de novo PD patients as disease controls.P = 0.004) and HAMD-17 scores (P = 0.001) than the HC group, and the ET group had higher HAMA scores (P = 0.011) and HAMD-17 scores (P = 0.011) than the HC group. The ET group had a significantly longer disease duration than the PD group (P < 0.001).The demographics and clinical characteristics of 37 ET, 37 PD and 35 HC are summarized in Table P = 0.023), acetic acid (P = 0.039), butyric acid (P = 0.020), isovaleric acid (P = 0.045) and isobutyric acid (P = 0.015). In further post hoc analysis, the ET group showed significantly lower levels of propionic acid (P = 0.023), butyric acid (P = 0.007) and isobutyric acid (P = 0.040) than the HC group. ET patients had lower levels of isovaleric acid (P = 0.014) and isobutyric acid (P = 0.005) than PD patients. Moreover, PD patients had lower fecal levels of propionic acid (P = 0.013), acetic acid (P = 0.016) and butyric acid (P = 0.041) than HCs . Differences between ET and HC in butyric acid and isobutyric acid were not significant but still had a trend (P = 0.082). ET patients had significantly decreased fecal levels of isobutyric acid compared with PD patients (P = 0.030). The difference between ET and PD in isovaleric acid was not significant but still had a trend (P = 0.084). PD patients were significantly lower in propionic acid (P = 0.023), acetic acid (P = 0.020), and butyric acid (P = 0.044) than HCs. These results  for further comparison  to distinguish ET patients from HCs. ET patients could be separated from HCs by butyric acid level with an AUC of 0.685 (95% CI: 0.556–0.814). Isobutyric acid level differences could discriminate ET patients from HCs with an AUC of 0.655 (95% CI: 0.525–0.786). By combining propionic acid, butyric acid and isobutyric acid levels, a higher AUC of 0.751 was obtained (95% CI: 0.634–0.867) with 74.3% sensitivity and 72.9% specificity Fig. .Fig. 2Dir = −0.349, P = 0.034), and fecal levels of isovaleric acid were negatively associated with FTM scores  and TETRAS scores . Among ET and PD patients, fecal levels of propionic acid were negatively correlated with SCOPA-AUT scores   or the PD group (P ≥ 0.246) . Among all the participants, fecal levels of propionic acid  and acetic acid  were both negatively correlated with Wexner scores , Lactobacillus , Catenibacterium , Howardella , Raoultella  and Candidatus Arthromitus  were found to be decreased in ET and positively correlated with the fecal levels of SCFAs. However, the abundance of Stenotrophomonas was increased in ET and negatively correlated with the fecal levels of isobutyric acid . After FDR adjustment, only the correlations between Faecalibacterium, Catenibacterium and SCFAs remained significant (P ≤ 0.045)  and isobutyric acid . After FDR adjustment, either of them remained significant (P ≥ 0.295)  with 74.3% sensitivity and 72.9% specificity, indicating their potential roles as diagnostic biomarkers for ET. Further analysis showed that fecal levels of propionic acid were negatively correlated with the Wexner score and SCOPA-AUT score. Isobutyric acid levels in feces were negatively associated with FTM scores. On the other hand, the lowered levels of butyric acid in ET were related to the decreased abundance of +-dependent or sodium-dependent monocarboxylate transporters. The absorbed SCFAs are used as a source of energy for colonocytes, and those that are not metabolized in colonocytes are transported into the portal circulation18. SCFAs can impact gut motility, strengthen gut barrier functions, and influence host metabolism and immunity19. The concentrations of fecal butyric acid, acetic acid and propionic acid were previously found to be decreased in PD patients compared with HCs17, which was consistent with our results. Our study identified reduced SCFAs in ET patients, but little is known about the role of SCFAs in the pathology of ET. Butyric acid and propionic acid could bind to GPCRs and influence GPCR-dependent signaling, such as MAPK and NF-κB signal transduction20. A leading concept of the gut–brain axis is that gut microbes secrete SCFAs that could influence host signaling, affecting functions in both the gut and brain. Because butyric acid and propionic acid exhibit strong inhibition of histone deacetylase (HDAC) activity21 and butyric acid might also act as a ligand of transcription factors, they have a broad impact on host metabolism, differentiation, and proliferation mainly due to their impact on gene regulation22. Based on evidence of SCFAs and neurodegenerative diseases, butyric acid has been implicated as a therapeutic candidate because of its ability to correct the disrupted activity of HDAC, which could mediate dopaminergic neuron death in PD25. Animal studies have also confirmed the capacity of butyric acid to prevent the degeneration of dopaminergic neurons and ameliorate motor impairments in PD models27. Propionic acid was found to limit inflammatory responses and protect BBB integrity29. Studies have shown that propionic acid promotes the survival of dopaminergic neurons against rotenone toxicity in the PD model30 and that orally administered propionic acid rescues dopaminergic neuronal loss and motor deficits in PD mice31. Little is known about the functions of isobutyric acid. However, a recent study reported that Bacteroides ovatus colonization in mice increased the abundance of intestinal SCFAs  and the concentrations of intestinal GABA, highlighting connections between gut microbiota and intestinal SCFA/neurotransmitter concentrations32. For ET, pathological changes in cerebellar abnormalities include changes to Purkinje cell axons and dendrites, displacement and loss of Purkinje cells, changes to basket cell axonal processes, abnormal distribution of climbing fiber connections to Purkinje cells and changes in GABA receptors in the dentate nucleus, resulting in reduced GABAergic output from the cerebellum33. Whether SCFAs are related to the neurodegeneration of Purkinje cells and the reduction in GABAergic output in the cerebellum remains unknown. Our results indicated a close relationship between SCFAs and ET; however, the cross-sectional study design precludes making any inferences of a causal relationship between SCFAs and the disease process in ET. Further longitudinal follow-up studies with serial measurements of fecal SCFAs as well as animal studies for mechanistic investigations are warranted.Most SCFAs produced in the colon are absorbed by colonocytes mainly via H34. The lack of SCFAs aggravated symptoms of constipation, and SCFA supplementation could attenuate constipation symptoms of PD35. Our results also demonstrated a significant association between the decrease in fecal SCFAs and the aggravation of constipation and autonomic dysfunction in ET patients. A case report found that microbiota transplantation simultaneously ameliorated patients’ essential tremor and irritable bowel syndrome7, which also suggested a close link between gut microbiota and ET. Thus, we considered that fecal SCFAs/microbiota could impact host gut motility and autonomic function.SCFAs are thought to stimulate colonic smooth muscle contractilityFaecalibacterium (correlated with butyric acid) and Catenibacterium . After FDR correction, the relationship remained significant. Both Faecalibacterium and Catenibacterium are SCFA-producing microbiota. Faecalibacterium is known to be the butyrate-producing microbiota36 and the major fermentation products of Catenibacterium are acetate, butyrate and lactate37. Faecalibacterium was 100% detected in both the ET and HC groups; the median relative abundance in the ET group was 2.06%, while the median relative abundance in the HC group was 3.28% (LDA 3.870). Catenibacterium was detected in 21.6% of the (8/37) HC group but was detected in only one sample of the ET group (1/35). The decrease in Catenibacterium and the undetectability of Catenibacterium in ET might also indicate its correlation to disease pathogenicity. The median relative abundance of Catenibacterium in the HC group was 0.07% (LDA 2.129). In addition, Lactobacillus was related to changes in fecal butyric acid , and Candidatus Arthromitus was associated with alterations in isobutyric acid . After FDR correction, only a trend of correlation remained without statistical significance. Lactobacillus is also considered an SCFA  producer38, while Candidatus Arthromitus is a specific inducer of differentiation in T helper 17 (Th17) cells, associated with the immune balance of Th1/2 and Treg/Th1739. A recent study demonstrated that the elevation of Candidatus Arthromitus in the feces might lead to colon inflammation, gut barrier dysfunction, and systemic inflammation40. Lachnoclostridium was increased in ET compared to PD. The abundance of Lachnoclostridium was found to be negatively correlated with isovaleric acid and isobutyric acid. After FDR adjustment, either of them remained significant (P ≥ 0.295). Lachnoclostridium is known to be an inflammation-associated bacterium that might lead to gut barrier dysfunction41. Our previous study reported gut microbiota changes in ET patients8. Here, we further reported SCFA alterations in ET and revealed the relationship between gut dysbiosis and SCFA changes. Decreased levels of SCFAs were closely related to gut dysbiosis and tremor severity in ET. Our results indicated that the gut–brain axis might have an important role in the pathogenesis of ET, but further studies of animal models are warranted.Lowered fecal levels of SCFAs in ET were found to be associated with decreased abundances of Catenibacterium. Little is known about the functions of isovaleric and isobutyric acid. A previous study indicated that Bacteroides ovatus colonization in mice increased the abundance of intestinal SCFAs  and the concentrations of intestinal GABA, highlighting links between gut microbiota and intestinal SCFA/neurotransmitter concentrations32. Interestingly, isobutyric acid was observed at similar levels between the PD and HC groups but was distinct between the ET and PD (or HC) groups. Isobutyric acid could distinguish ET from PD with an AUC of 0.718 (95% CI: 0.599–0.836) and identify ET from NC with an AUC of 0.655 (95% CI: 0.525–0.786). In addition, isobutyric acid levels were also related to tremor severity, which further strengthens its association with ET. Whether oral supplementation with isobutyric acid could relieve tremor severity in ET patients is worth future research.Compared with PD patients, ET patients had lower fecal levels of isovaleric acid and isobutyric acid. The combination of isovaleric acid and isobutyric acid identified ET from PD with an AUC of 0.743 (95% CI: 0.629–0.857) with 74.3% sensitivity and 62.9% specificity, indicating their potential roles as differential diagnostic biomarkers for ET. The levels of isovaleric acid in feces were negatively correlated with FTM scores and TETRAS scores. Isobutyric acid levels in feces were negatively associated with FTM scores. The lowered levels of isobutyric acid were related to the decreased abundance of In conclusion, fecal SCFAs were decreased in ET patients and correlated with the clinical severity and specific gut microbiota changes of ET. Fecal propionic acid, butyric acid and isobutyric acid might be diagnostic biomarkers for ET, while isobutyric acid and isovaleric acid might be differential diagnostic biomarkers for ET. Isobutyric acid changes in feces might be more ET-specific than other SCFA alterations.Our study has several limitations. First, dietary patterns and food preferences may impact microbiota expression, larger-scale research samples from different populations are warranted, and a complete and systematic dietary interrogation, such as the Food Frequency Questionnaire, should be introduced in future studies. Second, the cross-sectional study design precludes making any inferences of a causal relationship between SCFAs and the disease process in ET. Further longitudinal follow-up studies with serial measurements of fecal SCFAs are warranted. Third, the diagnostic and differential diagnostic abilities of fecal levels of SCFAs should be verified by independent samples from ET, HC and PD. Larger numbers of another independent fecal sample should be tested in the future. Finally, the PD patients in our cohort had a significantly shorter disease duration than the ET patients. We mainly matched ET, PD and HC in age, sex and BMI. Considering the difference in disease duration between the ET and PD groups, we also screened 33 early PD patients and 16 ET patients (disease duration ≤3 y) for further comparison. Between-group differences in SCFAs were generally consistent with our primary data. In addition, we did not find a correlation between disease duration and changes in SCFAs. However, it is better to recruit early PD and ET patients with short disease durations to complete the verification with a larger sample in the future.The research protocol was approved by the Ethics Committee of Ruijin Hospital affiliated with Shanghai Jiao Tong University School of Medicine (RHEC2018–243). Written informed consent was obtained from all of the participants.42 and PD diagnosis was based on MDS criteria43, (3) no anti-PD medication intake before the fecal sample collection of all subjects, (4) solely beta-blocker intake or no relevant medication intake before the fecal sample collection for ET group. HCs matched by age, sex, and body mass index (BMI) were selected simultaneously. Exclusion criteria were: (1) vegetarian, (2) malnutrition, (3) chronic gastrointestinal disorder , (4) severe chronic illness , (5) history of major gastrointestinal surgery, (6) ongoing or regular consumption of yogurt, (7) use of any probiotic or antibiotic within one month, (8) ongoing use of corticosteroid, proton pump inhibitor, statin, metformin, immunosuppressant or anti-neoplastic medication, and (9) severe cognitive deficit that obstructed the execution of clinical assessment.One hundred and nine subjects  from the outpatient clinic of the Movement Disorders Center in Ruijin Hospital affiliated with Shanghai Jiao Tong University School of Medicine were enrolled in our study between January 2019 and December 2022. Inclusion criteria were: (1) aged 25–85 years, (2) ET patients were diagnosed according to MDS Task Force criteria44 for anxiety, Hamilton Depression Scale-17 items (HAMD-17)45 for depression, Wexner constipation score46 and Bristol stool scale47 for constipation severity, and Mini Mental State Examination (MMSE)48 for cognition. The Scale for Outcomes in Parkinson’s Disease for Autonomic Symptoms (SCOPA-AUT)49 was interrogated for ET and PD patients for autonomic dysfunction. The Fahn-Tolosa-Marin Clinical Rating Scale for Tremor (FTM) and the Tremor Research Group (TRG) Essential Tremor Rating Assessment Scale (TETRAS)50 were examined among ET patients; the Movement Disorder Society sponsored version of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS)51 and the Hoehn and Yahr (H-Y)52 stage were examined for PD patients.All subjects provided information on medical history, weight, and height for the calculation of BMI and accepted neurological examination and clinical assessments, such as the Hamilton Anxiety Scale (HAMA)Each participant was asked to collect a fecal sample in the morning using fecal collection containers. The containers were transferred to ice and stored at −80 °C prior to processing. The analysis of SCFAs was performed following routine operations by Tinygene Bio-Tech (Shanghai) Co., Ltd. For each subject, 400 mg of fresh fecal samples was applied to the analysis of SCFAs after grinding and sonication pretreatment. Fecal analyses for individual SCFAs were performed with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography tandem MS (LC-MS/MS).The DNA was extracted from 200 mg samples using the QIAamp® Fast DNA Stool Mini Kit  following the manufacturer’s instructions. Microbial composition was determined by 16 S rRNA gene sequencing of DNA extracted from stool by amplifying the V3–V4 regions. DNA was checked by running the samples on 1.2% agarose gels. Polymerase chain reaction (PCR) amplification of 16 S rRNA genes was performed using general bacterial primers (357 F and 806 R) with a two-step amplicon library built on the Novaseq platform.t tests or analysis of variance (ANOVA) if normally distributed and with the nonparametric Mann–Whitney U test if assumptions of normality or homoscedasticity were violated. We used the area under the receiver operating characteristic (ROC) curve (AUC) to quantify the model’s diagnostic performance for exploring the ability of SCFAs to distinguish ET patients from HCs or PD patients. To examine the associations between SCFAs and clinical severity, we used Spearman correlation analysis. Statistical analysis was performed using SPSS software , and the significance levels, including P value and FDR-P, were set at 0.05 (two-tailed).Continuous variables are expressed as the mean ± standard deviation, and categorical variables are expressed as numbers and percentages. We tested the homogeneity of variances using Levene’s test. Variables were compared with two-tailed The 16 S sequences were analyzed by using a combination of software Trimmomatic (version 0.35), Flash (version 1.2.11), UPARSE (version v8.1.1756), mothur (version 1.33.3) and R (version 3.6.3). The raw 16 S rRNA gene data were processed to form operational taxonomic units (OTUs) at 97% identity using UPARSE. Taxonomy was assigned using Silva 128 as the reference database. The genus level of the relative abundance data was selected for further analysis. Linear discriminant analysis (LDA) effect size (LEfSE) analysis was used for between-group comparisons  with an alpha cutoff of 0.05 and an effect size cutoff of 2.0. The discriminant genera identified by LEfSE analysis were further used for the Spearman correlation analysis of SCFAs.Further information on research design is available in the Supplementary materialsReporting Summary


Capsicum annuum L.) is often affected by the critical salt stress. Salicylic acid (SA) can improve plants’ stress tolerance by promoting growth and regulating ion absorption and transportation.The growth and yield of pepper  contributing to alleviation of salt stress by SA in pepper. Besides photosynthesis related genes, GO and KEGG analyses revealed that the DEGs were enriched in ‘sequence-specific DNA binding’, ‘transcription regulator activity’ and ‘DNA binding transcription factor activity’ by GO terms. And our results showed that TFs, such as MYB, bZIP, BBX, AP2/ERF, NAC, etc., probably make a great contribution in the alleviation of salt stress by SA.To uncover the alleviated mechanism of salt stress by SA in pepper, we conducted morphological, physiological, cytological, and transcriptomic analyses under a single SA treatment and NaCl with and without SA pre-treatment for 9 days. Seedlings under NaCl treatment showed yellow shrunken leaves, this tatus were alleviated by NS treatment (NaCl with SA pre-treatment). Compared with plants under NaCl treatment, those in the NS treatment showed reduced lipid peroxidation, and significantly increased contents of chlorophyll and osmotic regulators . Treatment with SA balanced the NaThese results reveal that SA can improve plants’ stress tolerance by balancing ion absorption, gene expression and transcriptional regulation, which provide new ideas and resources for subsequent research on the mechanism of salt tolerance in pepper.The online version contains supplementary material available at 10.1007/s11033-022-08064-y. To keep the cytosolic Na+ at low level and maintain osmotic balance, plants employ different strategies to prevent and adapt to high Na+ concentrations: active Na+ efflux, Na+ compartmentalization in vacuoles, and prevention of Na+ influx [+ concentration in plant tissues can interfere with the function of K+. In plants, K+ participates in a series of metabolically related enzymatic reactions, and a high external Na+ concentration competes for K+ and disrupts cellular homeostasis [+ uptake and transport to control intracellular ion homeostasis in halophytes under high-salt conditions [+(K+)/H+ antiporters in plant cells are an important group of proteins that remove Na+ from the cytoplasm to maintain a low cytoplasmic Na+ concentration.Soil salinity is a major factor limiting plant productivity in many cultivated areas. More than 40% of irrigated agricultural land worldwide is predicted to be affected by salinity . At presrocesses . Maintence trait . Some plt stress . Thus, c+ influx . A high eostasis . Plant mnditions . The Na++ absorption [+/Na+ ratio maintaining in plants [Salicylic acid (SA) is an endogenous growth regulator  and belosorption , 20, caun plants . Thus, SPepper is one of the three most important Solanaceous vegetable crops worldwide, and it is generally considered to be salt sensitive . PeppersSA is important for plant growth and enhance stress tolerance, however, the expression of genes of the SA-mediated alleviation of salt stress, and how SA coordinate pepper salt stress acclimation at molecular levels is largely undisclosed. In this study, we treated pepper seedlings with NaCl, SA, or NS, or Hoagland’s nutrient solution (CK) for 9 days, then the morphological, physiological, and cytological analyses were investigated to compare the difference of the untreated or treated samples. We also performed RNA seq analyses on the leaves of these seedlings at 3 days under various treatments, the DEGs were thoroughly analysed to further explore the molecular mechanism of the alleviation of salt stress by SA.C. annuum L.) were surface sterilized using sodium hypochlorite  for 15 min, then washed with distilled water. The seeds were germinated in a Petri dish on filter paper, and the treatment liquid was changed every day. Healthy germinated seedlings were transferred into a seedling tray filled with peat, vermiculite, and perlite (vol/vol 1:1:0.5). Seedlings were grown in a growth chamber at 25/16 °C (16-h light/8-h dark photoperiod) with relative humidity of 70%. After 50 to 60 days, a hydroponic system was established using pepper seedlings with uniform growth. Four treatments were applied: CK (Hoagland’s nutrient solution), NaCl (100 mM), SA (0.15 mM), and NaCl + SA (NS) (SA: 0.15 mM + NaCl: 100 mM). The total treatment period was 9 days. Samples were taken for analysis after treated 3, 6, and 9 days. At each sampling time, samples from three replicates, three seedlings of each replicate were analysed simultaneously. A portion of each sample was frozen in liquid nitrogen and then stored frozen until further analyses.Healthy and viable seeds of pepper  to characterize stomatal morphology and density and to observe the arrangement of cells within the leaf. Samples were prepared for microscopy according to the method with minor modifications  and at l+ and K+ contents in leaves were determined as described elsewhere [Chlorophylls were extracted from leaf discs in 75% alcohol and stored in the dark for 24 h, the supernatant was measured at 646 nm and 663 nm, and the chlorophyll content was calculated according to Lichtenthaler . The levlsewhere Total RNAs were isolated from pepper leaves harvested on day 3 of the various treatments using an RNA Prep Pure Plant kit . The 12 RNA samples  were sent to Novogene-Bioinformatics Technology Co. Ltd.  for library construction and sequencing.The integrity of RNA was confirmed using an RNA Nano 6000 Assay kit and the Bioanalyzer 2100 system . Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® . Briefly, mRNA was purified from total RNA using polyA oligo-attached magnetic beads and fragmented using Illumina proprietary fragmentation buffer at 94 ℃ for 15 min. First-strand cDNA was synthesized using random hexamer primer and M-MLV Reverse Transcriptase (RNase-H). Second-strand cDNA was then synthesized. These cDNA fragments were then subjected to an end-repair process. The insert size of the library was restricted to approximately 250 ~ 300 bp using PCR products purified with the AMPure XP system .C. annuum. L_Zunla-1_v2.0. Hisat2 v2.0.5 was used to build the index for the reference genome and to align paired-end clean reads to the reference genome. Expression level of each gene was calculated by using reads per kb per million reads (RPKM) method [Raw data (raw reads) in fastq format were first processed using in-house perl scripts. Clean data (clean reads) were obtained by removing reads containing adapters, reads containing poly-N, and low-quality reads from the raw data. We then calculated the Q20, Q30, GC-content, and sequence duplication levels for the clean data. All the downstream analyses were based on high-quality clean data. The retained high-quality reads were mapped to the pepper reference genome ) method .To quantify the abundance of gene transcripts, all reads from samples were mapped onto the reference transcriptome by feature Counts (1.5.0-p3) . We usedP-value < 0.05 was indicative of a significantly enriched GO term. In the KEGG enrichment analysis, the cellular metabolic and biochemical pathways and potential biological behaviour of differentially expressed genes (DEGs) were examined. We used clusterProfiler R package to test the statistical enrichment of DEGs in KEGG pathways. A corrected P-value < 0.05 was set as the threshold to determine significant enrichment of gene sets.The GO enrichment analysis of DEGs was implemented using TBtools v1.06 , where aAfter 3-, 6-, and 9-day treatments, seedlings treated with SA and those in control showed vigorous growth. However, those plants treated with NaCl grown weakly resulting in curly and yellow leaves, while the seedlings in the NS treatment showed sturdier phenotypes similar to that of control Fig. A. This rTo further explore these morphological differences among treatments, we observed the ultrastructure of the young leaves. The leaf cells were tightly packed and the upper and lower epidermal cells were arranged regularly under SA treatment Fig. A. In conTo determine the physiological responses of pepper seedlings to salt and NS treatment, the proline contents, MDA contents, chlorophyll contents and soluble sugar contents were detected. With 3-, 6- and 9-day treatments, the proline contents of pepper leaves were much higher under the NaCl treatment and the NS treatment than that without any treatment. Treatment with SA alone resulted in a higher proline content than that of CK at day 6 and 9 but not at day 3 . We obtained approximately 73.24 million total reads, of which approximately 72.39 million passed the Illumina quality filtering threshold, yielding a quality rate of > 98.84%. This result indicated that the throughput sequencing data were sufficiently accurate to allow for further analyses.Reference-based transcriptome assemblies of RNA-seq data were performed using the reference pepper genome sequence. Mapping results showed that there were about 52,889,856 (95.13%), 58,029,558 (95.83%), 61,484,630 (95.97%), and 58,272,960 (95.36%) unigenes in the CK, NaCl, SA, and NS libraries, respectively (Table S2), corresponding to 90.21%, 91.21%, 90.72%, and 89.84% of reads mapped to the reference genome, respectively. These reads were then used for reference-guided assembly and differential expression analysis.To understand the mechanism of the alleviation of salt stress by SA in pepper, 901 related DEGs were selected by the intersection of NaCl vs. Ck DEGs and NS vs. NaCl DEGs Fig. A. In addWe performed a GO enrichment analysis to better understand the potential functions of DEGs involved in the alleviation of salt stress by SA in pepper. According to the results of sequence alignments, in total, there are 35 significant GO terms distributed in each three GO categories, namely molecular function (3 members), biological processes (12 members), and cellular components (20 members) . Among the significantly enriched pathways were ‘Photosynthesis—antenna proteins’, ‘Photosynthesis’, ‘Galactose metabolism’ and ‘Starch and sucrose metabolism’ Fig. . TherefoAs is known, TFs play an important part in tolerance of salt stress. We performed a global TF classification of the 901 DEGs to investigate the TFs function of alleviation of salt stress by SA in pepper. There were 66 TFs belonging to 25 TF families were among the DEGs. The TF families with the largest number and the highest proposition of DEGs were MYB , with AP2, bZIP , HB , NAC  and Orphans  as following . The MYB, AP2, bZIP and NAC TF families are known to play roles in plant defence responses . In addi+ absorption [+/Na+ ratio in plants [+ and Na+), and primary metabolome.In previous studies, the exogenous application of proline , nitric sorption , and capsorption , increasn plants . NeverthIn this study, the NaCl-treated pepper leaves were yellow and shrivelled, the root growth was weakened, showing that the plant growth was inhibited under salt stress Fig. . In our 2 concentration (Ci) were also dramatically affected by salinity and returned to a certain extent by SA treatment in pepper [In many studies, as another important aspect of plant growth, photosynthetic capacity is reduced under salt stress often attributed to inhibition of photosynthetic rate . Hence, n pepper . Reductin pepper . In addiIn our study, the lipid peroxidation of pepper leaves under salt stress were reduced after SA treatment, and the proline and sugar contents were also increased Fig. . Other s+ and K+ are the two main ions that play important role in cell homeostasis, and cells generally maintain high concentrations of K+ and low concentrations of Na+ [+ and K+ is even more important under salinity. Intracellular K+ and Na+ homeostasis has an important hold on enzymes activity, maintenance of membrane potential and osmotic potential of the cell for cell volume regulation and cell function [+ moving into plant cells and reaches toxic levels it disrupts enzymes and cell function [Nas of Na+ , 53. Homfunction , 53. Plafunction .+ accumulation, enhanced K+ absorption, and balance the Na+/K+ ratio ionic homeostasis [+, a higher Na+/K+ ratio, and decreased uptake of K+ were detected in pepper under salt stress treatment, while supplementation with SA alongside NaCl decreased Na+ accumulation and the Na+/K+ ratio, and enhanced the uptake of K+ . And the TFs among the DEGs with opposite expression pattern under salt without and with SA treatment, may be the key TFs involved in alleviation of salt stress by SA in pepper. In particular, the 317 DEGs (NaCl vs. CK_up and NS vs. NaCl_down) were significantly enriched in ‘sequence-specific DNA binding’, ‘transcription regulator activity’ and ‘DNA binding transcription factor activity’ by GO terms . Furthermore, we identified 42 TFs among these DEGs, as the predominance in all of the DEGs (Table S3). These results indicate that the different expressed TFs under salt stress treatment and down-regulated by salt with SA treatment play an essential role in alleviation of salt stress by SA in pepper.VvMYB62 in Arabidopsis was shown to increase AtNHX expression, and increase the germination rate and salt tolerance of transgenic plants [Capana04g000509, Capana03g000766, Capana12g002172, Capana10g001433 and Capana06g002789 were induced to salt stress and inhibited by salt with SA, as well as five bZIP TF genes . MYB may play a role in SA-mediated salt response resulting in MYB expressed alterably under salt stress with SA [HB  genes were identified among these different expressed TF genes. BBX TFs are well-known to be involved in plant development, especially light, circadian signalling and flowering in plant [BBX genes also found to be in these 44 TFs, and all these four BBX genes were induced expressed under salt stress condition, down regulated by salt with SA treatment. This indicated that BBX TFs are take part in alleviation of salt stress by SA. AP2-EREBP TFs are a kind of plant-specific TFs which have been identified 175 members in pepper [Among these 42 TFs, there are five MYB TFs with proportion of 12%, as well as bZIP, were identified as the regulator involved in alleviation of salt stress by salicylic acid in pepper . MYB is a super family of transcription factor, which play essential roles in plant growth and development, primary and secondary metabolism, stress response and so on . Over-exc plants . In our  with SA , 62, and with SA . Furtherin plant . And ourSalt stress causes diverse damage on pepper plant, leading to production and value loss. Several previous studies reported that the alleviation of salt stress by SA in several plants physiologically. However, the molecular mechanism of the alleviation of salt stress by SA are still rarely understood. We have integrated morphological, physiological, cytological, and transcriptomic data to explore the role of SA mitigating salt stress in pepper. We identified a number of DEGs and annotated them using the GO and KEGG databases. The results show that important DEGs which expressed induced expressed under salt stress condition, down regulated by salt with SA treatment, are enriched in ‘sequence-specific DNA binding’, ‘transcription regulator activity’ and ‘DNA binding transcription factor activity’ by GO terms. This indicated that TFs probably make a great contribution in the alleviation of salt stress by SA. Taken together, these data shed light on the molecular mechanism of TFs in plants, and provide important clues for further research on genes and networks that function to confer salt tolerance in pepper. We can also conclude that transcriptomics-assisted crop trait studies are a useful tool for studying the regulatory mechanisms of stress tolerance.Supplementary file1 (Transcription factor families distribution of 901 identified DEGs.)Supplementary file2 , biological process (BP), and cellular component (CC). X-axis indicates GO term. Y-axis represents the level of significance of GO term enrichment.)Supplementary file3 (Transcription factor families’ distribution of identified DEGs belonged to NaCl vs. CK_up and NS vs. NaCl_down section.)Supplementary file4 (XLSX 2422 KB)Supplementary file5 (DOCX 14 KB)Below is the link to the electronic supplementary material.


The relation of social deprivation with single cardiometabolic disease (CMD) was widely investigated, whereas the association with cardiometabolic multi-morbidity (CMM), defined as experiencing more than two CMDs during the lifetime, is poorly understood.We analyzed 345,417 UK Biobank participants without any CMDs at recruitment to study the relation between social deprivation and four CMDs including type II diabetes (T2D), coronary artery disease (CAD), stroke and hypertension. Social deprivation was measured by Townsend deprivation index (TDI), and CMM was defined as occurrence of two or more of the above four diseases. Multivariable Cox models were performed to estimate hazard ratios (HRs) per one standard deviation (SD) change and in quartile , as well as 95% confidence intervals (95% CIs).During the follow up, 68,338 participants developed at least one CMD (median follow up of 13.2 years), 16,225 further developed CMM (median follow up of 13.4 years), and 18,876 ultimately died from all causes (median follow up of 13.4 years). Compared to Q1 of TDI (lowest deprivation), the multivariable adjusted HR (95%CIs) of Q4 (highest deprivation) among participants free of any CMDs was 1.23 (1.20 ~ 1.26) for developing one CMD, 1.42 (1.35 ~ 1.48) for developing CMM, and 1.34 (1.27 ~ 1.41) for all-cause mortality. Among participants with one CMD, the adjusted HR (95%CIs) of Q4 was 1.30 (1.27 ~ 1.33) for developing CMM and 1.34 (1.27 ~ 1.41) for all-cause mortality, with HR (95%CIs) = 1.11 (1.06 ~ 1.16) for T2D patients, 1.07 (1.03 ~ 1.11) for CAD patients, 1.07 (1.00 ~ 1.15) for stroke patients, and 1.24 (1.21 ~ 1.28) for hypertension patients. Among participants with CMM, TDI was also related to the risk of all-cause mortality .We revealed that people living with high deprived conditions would suffer from higher hazard of CMD, CMM and all-cause mortality.The online version contains supplementary material available at 10.1186/s12889-023-17008-5. Cardiometabolic diseases (CMDs) are the leading cause of premature mortality worldwide and produce extremely high socioeconomic burden on healthcare resources . We hereAlthough it has never stopped finding out the biological and clinical mechanism of CMDs, there is a growing awareness that socioeconomic factors also play an important role in the course of these diseases and that more considerate and broad prevention strategies are urgently needed , 10. AmoThe association between high socioeconomic deprivation and increased risk of new-onset CMD and mortality has been well documented in literature. For example, elevated socioeconomic deprivation was shown to associate with greater risk of T2D, hospitalization, and related mortality –19. A coUnderstanding the intricate association between social deprivation and the whole course from CMD to CMM or to mortality can provide insightful evidence linking social inequality and CMM/death progression trajectory. To our knowledge, the relation between social deprivation and CMM and CMM-related mortality has not been completely explored thus far. To fill this gap, in the present work we aim to perform a comprehensive exploration to assess whether and to what extent social deprivation is associated with CMD, CMM, and mortality under different baseline conditions using data source of over 0.5 million UK Biobank participants . FurtherN = 1,298), (2) had missing data in TDI (N = 624), (3) suffered from type I diabetes  or gestational diabetes , and (4) suffered from any of CMDs  before recruitment. Finally, 345,417 “healthy” individuals without any CMD at recruitment were included in our subsequent analyses  which derived from the postcode of residence by aggregating data of unemployment, car and home ownership, and household overcrowding .The four CMDs were mainly ascertained via ICD-10 codes , the Scottish Morbidity Records (Scotland), and the Patient Episode Database . CMM was defined as the situation where a patient suffered from at least two types of the four diseases, and CMM onset was determined as the date when the second disease was diagnosed. All-cause mortality and death date were obtained through death certificates held by the National Health Service (NHS) Information Centre  and the NHS Central Register (Scotland).In our UK Biobank cohort, the complete date of follow-up was 19 July 2022; we employed it as the censoring date or used it to calculate survival time .Several risk covariates were taken into account, including age (Field 21022), sex (Field 31), ethnicity (Field 21000), education (Field 6138), income (Field 738), former smoking status (Fields 1249 and 20160), former drinking status (Fields 3731 and 20117), physical activity (Field 22032), healthy diet score , body mass index (BMI) (Field 21001), systolic blood pressure (SBP) (Fields 93 and 4080), and diastolic blood pressure (DBP) (Fields 94 and 4079). For covariates with multiple measurements, we calculated the average for continuous variables, and screened across all measurements to examine the presence for categorical variables, considering simultaneously the use of as much data as possible and time sequence of causality. For example, we calculated the average for four times assessment of BMI. In addition, if there had a former smoking history at any measurement for a given participant, we considered this participant to be a former smoker although she/he might be reported as a former non-smoking at the initial visit. For cases, only assessment visit before onset of disease was taken into account. More details regarding the definition and coding of these covariates are described in the Supplemental File.Survival time of each participant was calculated as the duration from the baseline to the date of diagnosis, death, or censoring 19 July 2022), whichever came first. The restricted cubic spline curve  was draw022, whicN = 345,417), we examined the association of TDI with onset of single CMD and CMM and all-cause mortality; for those participants diagnosed as at least one CMD , we estimated the association of TDI with CMM onset and all-cause mortality; for participants suffering from CMM , we evaluated the association of TDI with all-cause mortality. The PH assumption was evaluated via the method given in [P > 0.05). The hazard ratio (HR) per one standard deviation (SD) change or in quartile and its 95% confidence intervals (CIs) were reported. All analyses were performed within the R software environment (version 4.1.3). The P value was two-sided, with the statistical significance threshold set to 0.05.The associations of TDI with morbidity and mortality of these diseases were evaluated by multivariable Cox proportional hazards (PH) model. For participants free of any CMDs at recruitment  was implemented to impute missing value of each covariate . We gene2, defined by WHO [Considering that the association pattern between TDI and CMD likely differed by distinct levels of covariates such as gender , 30 and d by WHO ).N = 14,866) [N = 38) [Further, we performed several sensitivity analyses: (1) only in participants of white ethnicity; (2) excluding case/death events occurring within the first two years of follow-up to reduce potential reverse causation; (3) re-ascertaining the outcomes only using ICD-10 codes; (4) excluding individuals with cancer   or all-c[N = 38)  at recruPe denoted as the exposure prevalence with the highest quartile of TDI. The 95% CIs of the AR% and PAR% was calculated using a plug-in method [Attributable risk percent (AR%) and population attributable risk percent (PAR%) were calculated to estimate the percentage of decreased cases/deaths if reducing TDI in the population with high TDI . The calculation formulas weren method .The analyses were based on existing data in UK Biobank. To our knowledge, no patients were involved in the design, recruitment, or conduct of the studies, nor did they participate in research question and outcome measures. The study results could not be disseminated to each participant due to deidentification; however, they could obtain the results through broadcasts or science articles.During a median follow-up of 13.2 years, a total of 68,338 participants developed at least one CMD among those without any CMD at recruitment. Among those participants, 9,210 participants developed T2D (median follow up of 13.4 years), 18,645 developed CAD (median follow up of 13.4 years), 5,698 developed strokes (median follow up of 13.2 years), and 53,093 developed hypertensions (median follow up of 13.3 years). The number of participants for each CMD alone and different statuses of CMM was shown in Fig. The baseline characteristics of all included participants under different conditions are summarized in Table For participants without any CMD at baseline, after the adjustment of covariates, TDI was significantly related to elevated risk of developing one CMD  Table , with HRFor participants without any CMD at baseline, after adjusting for covariates, we discovered that per 1 SD increase of TDI could lead to 16% (95%CIs = 14% ~ 18%) higher risk of developing CMM or 15% (95%CIs = 13% ~ 17%) larger risk of all-cause mortality, respectively, with the HR of Q4 being 1.42 (95%CIs = 1.35 ~ 1.48) and 1.34 (95%CIs = 1.27 ~ 1.4122), respectively. Meanwhile, an incremental trend of HR from Q1 to Q4 was observed for each association scenario  higher risk of CMM (median follow up of 4.8 years) and 14% (95%CIs = 12% ~ 16%) higher risk of all-cause mortality (median follow up of 5.4 years) Table .Again, for each CMD, compared to participants with Q1 of TDI, the HR of participants with Q4 for developing CMM or all-cause death was much larger than that of TDI when it was treated as a continuous variable, and an increased trend of HR from Q1 to Q4 was seen for every disease Table . In addiDuring the median follow up of 4.5 years, after adjusting for covariates, we identified that per 1 SD increase of TDI could result in 13% (95%CIs = 11% ~ 15%) higher risk of death among participants with CMM, with the HR of Q4 equal to 1.35 (95%CIs = 1.28 ~ 1.43) compared to Q1  to 37.5% , and the PAR% ranged from 1.7% (from CAD baseline to CMM or from stroke baseline to CMM) to 13.0%   [Our current findings of association between TDI and individual CMDs were in agreement with previous research , 23, 29. = 1.24) .Besides revealing that TDI was an independent risk factor of CMM and all-cause mortality, our work is also of sociological importance. It has been well-documented that socioeconomic disadvantage has a pronounced influence on human health outcomes –50, and The major strength of our study is that large-scale samples and long follow-up periods enabled us to evaluate the role of TDI systematically and thoroughly during the whole progress from baseline to CMM and death. In addition, our work was among the first to survey the role of TDI in the progression trajectory of CMM.There are several limitations to this study. First, our findings could be vulnerable to selection bias, which has been extensively discussed in previous papers –56. It hSecond, although we made efforts to control for various common potential confounders associated with cardiovascular disease and diabetes, it is essential to recognize that there might be other residual and unmeasured confounders that were not accounted for but would affect the disease outcomes in our study , such asThird, TDI was generated from aggregated data related to the postal region, which is not measured at the individual level and may not completely reflect the differences in actual individual socioeconomic deprivation , 59. On Fourth, non-CMD comorbidities such as cancer and dementia were not adjusted for in our models, however, these co-existing conditions could confound the results , 65. To Finally, our study was conducted as an observational population study, which limited our ability to make a conclusion about the causality between TDI and CMM. Further studies with multiple design strategies are required to confirm our findings and provide a more comprehensive understanding of the potential mechanism underlying the associations observed in the present study.We provided epidemiological evidence about the adverse influence of TDI on each CMD, and further revealed that people with one CMD would suffer from higher hazard of CMM and all-cause mortality, and that people with CMM would encounter increased risk of all-cause mortality if living with high deprived conditions.Additional file 1: Supplementary information 1. Treatment for covariates. Supplementary Table S1. Definitions of the four cardiometabolic diseases. Supplementary Table S2. Attributable risk percent (AR%) and population attributable risk percent (PAR%) of one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S1. Restricted cubic spline (RCS) curves for the association of TDI with one CMD, CMM and all-cause mortality. Supplementary Figure S2. Cumulative risk curves for the association of TDI with one CMD, CMM and all-cause mortality. Supplementary Figure S3. Correlation coefficients between TDI and other covariates. Supplementary Figure S4. Subgroup analyses by sex group for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S5. Subgroup analyses by age group for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S6. Subgroup analyses by income group for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S7. Subgroup analyses by education background for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S8. Subgroup analyses by smoking status for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S9. Subgroup analyses by drinking status for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S10. Subgroup analyses by physical activity for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S11. Subgroup analyses by healthy diet score for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S12. Subgroup analyses by BMI for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S13. Analyses in white ethnicity for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions. Supplementary Figure S14. Sensitivity analyses for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions while excluding events occurred within the first two years of follow-up. Supplementary Figure S15. Sensitivity analyses for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions only according to ICD-10 codes. Supplementary Figure S16. Sensitivity analyses for the association of Townsend deprivation index with one CMD, CMM and all-cause mortality from different baseline conditions while excluding individuals with cancer or dementia at recruitment.


Sevoflurane anesthesia is widely used in pediatric ambulatory surgery. However, emergency agitation (EA) and emergency delirium (ED), as major complications following sevoflurane anesthesia in children, pose risks to surgery and prognosis. Identifying the high risk of EA/ED, especially anesthesia exposure and the depth of anesthesia, may allow preemptive treatment.LOW−BIS/TimeANES %, EtSevo (%) and EtSevo-time AUC were included in the multivariate logistic regression model as independent variables to analyze their association with EA or ED.A total of 137 patients were prospectively enrolled in this single-center observational cohort study to assess the incidence of EA or ED. Multivariable logistic regression analyses were used to test the association between volatile anesthesia exposure and depth with EA or ED. The Richmond Agitation and Sedation Scale (RASS), Pediatric Anesthesia Emergence Delirium Scale (PAED) and Face, Legs, Activity, Cry, and Consolability (FLACC) behavioural pain scale was used to assess the severity of EA or ED severity and pain. Bispectral index (BIS) to monitor the depth of anesthesia, as well as Timep = 0.034], peak FLACC [2.56  p < 0.001] and adverse event (respiratory complications) [0.03  p = 0.003] were independently associated with higher odds of EA. Taking EtSevo-time AUC ≤ 2,000 as a reference, the incidence of EA were [15.84  p = 0.002] times and 16.59  p = 0.009] times for EtSevo-time AUC 2,500–3,000 and EtSevo-time AUC > 3,000, respectively. Peak FLACC [3.46  p < 0.001] and intraoperative agitation [5.61  p = 0.001] were independently associated with higher odds of developing ED. EtSevo (%), intraoperative BIS value and the percentage of the duration of anesthesia at different depths of anesthesia  were not associated with EA and ED.The overall prevalence of EA and ED was 73/137 (53.3%) and 75/137 (54.7%) respectively, where 48/137 (35.0%), 19/137 (13.9%), and 6/137 (4.4%) had mild, moderate, and severe EA. When the recovery period was lengthened, the prevalence of ED and extent of FLACC decreased and finally normalized within 30 min in recovered period. Multivariable logistic regression demonstrated that intraoperative agitation [2.84  For pediatrics undergoing ambulatory surgery where sevoflurane anesthesia was administered, EA was associated with surgical time, peak FLACC, respiratory complications, and “EtSevo-time AUC” with a dose-response relationship; ED was associated with peak FLACC and intraoperative agitation. Sevoflurane anesthesia has advantages of rapid uptake and elimination and shows reduced opioid adverse events in pediatric ambulatory surgery –3. HowevSeveral perioperative factors may be associated with EA or ED. Volatile anesthesia exposure and depth are the major risk factors for EA/ED . VolatilThe bispectral index (BIS) monitor is a common method for monitoring the depth of anesthesia , 11. PreTo explore the relationship between depth of anesthesia or volatile exposure and EA or ED, we conducted a prospective observational cohort study that explored independent association between sevoflurane exposure and low BIS with EA/ED in children with sevoflurane anesthesia undergoing ambulatory surgery. We also explored the utility of the age, preoperative modified Yale Preoperative Anxiety Scale (m-YPAS), anesthesia time, surgical time, the post-operative peak scores of Face, Legs, Activity, Cry, and Consolability (peak FLACC), intraoperative agitation and adverse event (respiratory complications) for EA or ED prediction.This present study was conducted at a single institution . The study was approved by the Affiliated Hospital of Zunyi Medical University Biomedical Research Ethics Committees (reference number: KLL-2022-623) and registered with the Chinese Clinical Trial Registry (ChiCTR2200062680). Recruitment occurred between August 15 and October 15, 2022. Written informed consent was obtained from child's parents. This study was consistent with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guidelines .The inclusion criteria were as follows: Children aged 4–12 years scheduled for same-day or ambulatory circumcision treatment under general anesthesia and American Society of Anesthesiology (ASA) physical status I–II. Exclusion criteria were developmental delay or neurologic impairment; autism spectrum disorder (ASD) or reactive attachment disorder (RAD); premedication interfering with the central nervous system; history of allergies, or adverse reactions to intraoperative medications. The diagnoses of exclusion were verified by the description of the children's parents, or diagnosis by a paediatrician. Developmental delay is defined as significantly delays in cognitive and physical development of children, including motor adaptive skills, language or personal and social behavior . NeuroloAfter screening for eligibility, informed consent for processing of personal data from the guardian was obtained at the time of the patient's appointment for surgery. On the day of surgery, after separation from the parents, the patients were assessed for preoperative anxiety by the m-YPAS  in the ovia inhalation of sevoflurane through a mask with the patient's consent and cooperation. A dorsal penile nerve block with 0.25% ropivacaine and 1% lidocaine compound (0.15 ml/kg) was administered following induction. Two pediatric urologists on the same team performed the circumcision (sutureless prepuceplasty). Volatile dose, airway management, management for any arising complications were administered at the discretion of the anesthesiologist.Volatile induction and maintenance anesthesia (VIMA) was performed After completion of the procedure, the patient was transferred to PACU for recovery and assessment of the level of agitation and delirium, the full recovery routine usually lasts 30 min in PACU and the patients are returned to the pediatric day ward when awake. Patients were discharged when the Modified Aldrete Score was ≥9 .•Pre-operation: age, height, weight, body mass index (BMI), and ASA class. Preoperative anxiety was assessed using the m-YPAS after separation from the parents, which included four items: activity, emotional expressivity, state of arousal, vocalization.•Surgical data included the type of procedure and duration of surgery (from incision to the end of surgery).•2), electrocardiogram (ECG), end-tidal sevoflurane concentration (EtSevo) with once every 15 s, temperature, end-tidal CO2 concentration (EtCO2). Other data included anesthesia duration , intraoperative agitation (nonpurposeful movement during anesthesia), and respiratory complications such as upper airway obstruction, hypoxia, bronchospasm, and cough, which were recorded in operation room and PACU.Anesthesia data: routine monitoring included noninvasive blood pressure (NBP), heart rate (HR), pulse oximetry  −1 or 0  . The recThe RASS is recognized as the most commonly used sedation and agitation assessment tool in the PACU or intensive care unit (ICU) . We asseThe PAED  and FLACLOW−BIS/TimeANES % and EtSevo-time AUC. BIS data and EtSevo(%) were fitted to the exposure duration to further detected the association between depth of anesthesia or sevoflurane exposure and EA/ED.•LOW−BIS/TimeANES %TimeLOW−BIS/TimeANES %included TimeBIS ≤ 40/TimeANES %, TimeBIS ≤ 30/TimeANES % and TimeBIS ≤ 20/TimeANES %, which were respectively expressed as the percentage of the duration of BIS ≤ 40, BIS ≤ 30, BIS ≤ 20 in the total anesthesia duration.Time•EtSevo-time AUCX-axis and EtSevo (%) as the Y-axis , which is a pharmacokinetic parameter and represents the plasma drug concentration-time curve. Sevoflurane concentrations were extracted at 5 min intervals during anesthetic exposure as the drug concentration at discrete points in time, using GraphPad PRISM software to calculate definite integral of the concentration of EtSevo as a function of exposure time with time-point as the e Y-axis . This reWe derived the acquisitive data and calculated TimeZ test with unpooled variance is used to test the statistics. The significance level of the test was set at p < 0.05. The final target of evaluable cases was 137 to account for a 20% attrition rate related to protocol violations.Based on sample size estimates from previous literature, the prevalence of EA or ED in children under sevoflurane anesthesia is approximately 60%–70% , 22. GroContinuous variables were summarized by medians and interquartile ranges due to nonnormality. Categorical variables were expressed as counts and percentages. The primary outcomes were EA and ED. Wilcoxon rank sum test, chi-square test, or Fisher exact test, as appropriate, were used compared the differences between continuous or categorical variables and between the two outcome indicators.p < 0.05 was statistically significant. We used IBM SPSS Statistics, version 25 (IBM Corp) and GraphPad PRISM (GraphPad Software) for statistical analyses.A univariate logistic regression model was established to calculate the crude odds ratio (OR) of each variable. Two multivariable logistic regression models were performed to assess the association between EA or ED and 6 independent variables: EtSevo-time AUC, BIS, surgical time, peak FLACC, intraoperative agitation, adverse event (respiratory complications). The variance inflation factor (VIF) measures the degree of multicollinearity or collinearity in the regression model, A total of 196 patients were recruited between August 15 and October15, 2022. We excluded 73 patients because they did not complete day-surgery and observations, or had events that might interfere with EA and ED. Ultimately a total of 137 patients were included in the analyses .2 and 29.2 . The median BIS value was 38.6 , and BIS > 40 and BIS ≤ 40 were 57/137 (41.6%) and 80/137 (58.4%), respectively. The percentage of the duration of BIS ≤ 40, BIS ≤ 30, BIS ≤ 20 in the total anesthesia duration were 84.7% , 35.3% , 9.4% , respectively. The median EtSevo and EtSevo-time AUC were 4.4%  and 2,618.0 , and the EtSevo-time AUC was calculated using software  and 75/137 (54.7%) respectively, with both EA and ED occurring in 59/137(43.1%) patients. The observed prevalence of different grades of EA was mild 48/137 (35.0%), moderate 19/137 (13.9%), and severe 6/137 (4.4%), and the median agitation duration was 1  min. Patients displaying a severe grade and frequent agitation lasting ≥3 min were less frequent. The prevalence of ED and assessment of FLACC decreased when the recovery period was prolonged, and finally disappeared within 30 min in the recovered period.p < 0.05. Intraoperative agitation and peak FLACC were associated with ED. EtSevo, intraoperative BIS value and the percentage of the duration for different depths of anesthesia  in the total duration of anesthesia were not associated with EA and ED. We made two univariate logistic regression models  p = 0.034], peak FLACC[2.56  p < 0.001] and adverse event (respiratory complications) [0.03  p = 0.003] were independently associated with higher odds of EA. Taking the EtSevo-time AUC ≤ 2,000 as a reference, the incidence of EA were [15.84  p = 0.002] times and 16.59  p = 0.009] times for EtSevo-time AUC 2,500–3,000 and EtSevo-time AUC > 3,000, respectively  p = 0.001] were independently associated with higher odds of ED.Based on univariate analysis, EtSevo-time AUC, intraoperative agitation, surgical time and post-operative peak FLACC were associated with EA; unadjusted n models . The outn models  and ED  EtSevo-time AUC fitted by EtSevo (%) and exposure time is independent risk factor for EA. Further it was found the risk factors had a dose-dependent effect, but not the two parameters alone. (2) Using BIS to assess the depth of anesthesia, it was found that there was no correlation with EA/ED. Similarly duration of low-normal anesthesia had not correlation with the occurrence of EA/ED (3) only 4.4% patients had severe EA/ED, and the median PAED score fell from 10 to 0 within 30 min of recover from anesthesia.In this study, we found that EtSevo-time AUC formed by fitting EtSevo (%) and exposure time was the main factor influencing EA, although EtSevo (%) and exposure time were not the independent factor. Meanwhile, EtSevo-time AUC trend to have a continuous dose-dependent effect on EA risk. AUC is recommended as the primary measurement with drug absorption data by The Food and Drug Administration (FDA) , and is The incidence of EA in patients in the prospective cohort was 20.0% with EtSevo-time AUC ≤ 2,000, 43.5% with EtSevo-time AUC 2,000–2,500, 54.3% with EtSevo-time AUC 2,500–3,000, 69.6% with EtSevo-time AUC > 3,000. It is suggested that anesthesiologists should pay attention to concentration of sevoflurane as well as exposure time. Excessive exposure may increase the accumulation of compounds in the body, which are more difficult to cleared during emergence , 27, resThe effect of depth of anesthesia on ED remains uncertain. In the study, we obtained the BIS value every 15 s during anesthesia for each patient, and found that the median BIS was not associated with EA or ED. We further found that the percentage of the duration of BIS ≤ 40, BIS ≤ 30, BIS ≤ 20 in the total anesthesia time was no correlation with EA or ED. This suggested that neither the depth of anesthesia nor the duration of low-normal anesthesia is an independent risk factor for EA or ED with transient exposure to sevoflurane. This result is similar to previous studies , 32. IntIn this study, we distinguished between EA and ED. EA is a common symptom during recovery from general anesthesia and has been described as an unpleasant state of extreme arousal that emergence alone and does not always follow delirium. ED is defined as acute brain dysfunction accompanied by mental confusion, agitation, and disinhibition during recovery from general anesthesia . The PAEThis study showed that children undergoing ambulatory surgery with sevoflurane anesthesia had a high incidence of EA or ED, but most had mild to moderate agitation, and only 4.4% had severe agitation. Delirium occurred within 30 min of recover from anesthesia and returned to normal before PACU discharge (within 30 min after recovery), consisting with other similar studies .The risk factors for EA/ED are presented in three major categories: patient-related, anesthesia-related, and surgical factors. We collected data on preoperative anxious, surgical duration, peak value of FLACC, and respiratory complications , the result shows that preoperative anxious, surgical duration were not associated with EA or ED. The independent risk factors for EA and ED were peak value of FLACC, intraoperative agitation and respiratory complications. Postoperative pain and intraoperative agitation have been clearly identified as risk factor for EA or ED. When the analgesic effect was poor, the rates of EA and ED increased by 1.86 times and 2.46 times . In thisThis study has some limitations. Firstly, the single center of the study, limitation to one surgical procedure, and anesthetic method in this study only represented the observation results for pediatric ambulatory surgery undergoing sevoflurane anesthesia. Due to this limited sample, the EA- or ED-related findings are not directly translatable to the general population. Multi-center trials might refine the interpretation of this trial. Secondly, the BIS data, which measures the level of consciousness under anesthesia, has proved controversial. This study should be repeated with other assessments of the depth of anesthesia. Thirdly, the psychiatric symptoms of children after discharge were not described. Fourthly, we did not assess the impact of hypoactive delirium on outcomes following general anesthesia, this aspects needs more attention.We found that using sevoflurane anesthesia in pediatric patients undergoing ambulatory surgery, the incidence of EA was associated with the fitted value “EtSevo-time AUC”, with a dose-related response, but not with depth of anesthesia as assessed by BIS and the duration of low-normal anesthesia. The fit of inhalation concentration and duration in sevoflurane anesthesia is a better indicator of anesthesia exposure than a single element. Further studies are warranted to validate our results and gain insight into the possible explanations for such an association.


Neurosurgical resection is a standard local treatment for lung cancer brain metastases (BMs). This study aims to investigate whether neurosurgical resection provides survival benefit in lung cancer BMs with poor KPS.This multicenter retrospective study included 386 lung cancer BMs with pretreatment KPS ≤ 70 among a total of 1177 lung cancer BMs treated at three centers from August 2010 to July 2021. Data analysis was performed from July to September 2022. Inverse probability of treatment weighting (IPTW) and propensity scores matching (PSM) based on propensity scoring were used to minimize bias. The main outcome was overall survival (OS) after diagnosis of BMs. Risk factors of OS were estimated using Cox proportional hazards regression models. All Characteristics were included in the multivariate Cox regression.P = 0.01), and PSM-adjusted multivariate COX analysis , IPTW-adjusted multivariate COX analysis . OS was significantly longer in neurosurgical resection group compared with non-surgical resection group according to unadjusted data , PSM-adjusted data  and IPTW-adjusted data .386 patients with pretreatment KPS ≤ 70 were included . Among them, 111 patients received neurosurgical resection, while 275 patients did not. Baseline characteristics were balanced between groups after IPTW or PSM. Neurosurgical resection was associated with significantly better prognosis in unadjusted multivariate COX analysis (hazard ratio [HR]: 0.68, 95% confidence interval [CI]: 0.51–0.91, Neurosurgical resection was associated with improved survival in patients with lung cancer BMs with poor KPS, suggesting that poor KPS is not a contraindication for neurosurgical resection in these patients.The online version contains supplementary material available at 10.1007/s11060-023-04293-8. Lung cancer accounts for up to 56% of all brain metastases (BMs), with lung cancer being the most common primary cancer in BMs. Lung cancer brain metastasis is associated with high morbidity and limited survival , and braKPS has been widely used to assess the activity, work, and self-care abilities of cancer patients for decades . Several. and White et al. presented retrospective clinical studies regarding the surgical resection of BMs [Surgical resection has become a standard local treatment for BMs and has demonstrated survival benefits in patients with large and single BMs . In the n of BMs , 14, whin of BMs , 15. In n of BMs  We consiSurgical resection of BMs is traditionally carried out in patients with good KPS –18; howehttps://clinicaltrials.gov/).This multicenter, retrospective cohort study included 386 patients with lung cancer BMs with pretreatment KPS ≤ 70, among a total of 1177 patients with lung cancer BMs. All patients were treated at two tertiary cancer centers and one tertiary comprehensive hospital in southern China between August 10, 2010 and July 1, 2021. The study was approved by the institutional review board of Sun Yat-sen University Cancer Center, which waived the need for informed consent. This study has been reported in line with the STROCSS criteria  and was The inclusion criteria were: (1) pathological evidence of primary lung cancer; (2) BMs confirmed by enhanced MRI; and (3) availability of complete clinical information. The exclusion criteria were: (1) patients with two or more types of cancer; (2) overall survival (OS) < 1 month; (3) patients receiving ventricle puncture surgery or other non-surgical resection; and (4) multiple encounters for the same patient. Data for the initial visit were used in the analysis for patients with multiple visits. Furthermore, we considered that 1 month was too short as an exposure period, and we therefore excluded patients with an OS time of < 1 month. Among all the patients with lung cancer BMs, patients with pretreatment KPS ≤ 70 were finally selected and divided into a surgical resection group (surgery group) and a non-surgical resection group (non-surgery group). We defined a pretreatment KPS ≤ 70 as a poor KPS because such a score was associated with poor outcomes in patients with lung cancer BMs in a previous study .The main outcome was OS after diagnosis of BM. OS was defined as the time from the date of BM diagnosis to the date of last follow-up or date of death or censoring. Follow-up data were collected at clinical visits and by telephone consultations.EGFR)/anaplastic lymphoma kinase (ALK) status, extracranial metastases, synchronous metastases, location of BMs, number of BMs, radiotherapy , chemotherapy and target therapy or immunotherapy were collected from the hospital information system and medical records.Baseline characteristics including age, sex, smoking history, histology, epidermal growth factor receptor -based adjustment methods: propensity score matching (PSM) and inverse probability of treatment weighting (IPTW) , 21. The2 tests for categorical variables. Differences in OS were compared using Kaplan–Meier analysis and the log-rank test. Risk factors for OS were evaluated by univariate and multivariate Cox proportional hazards regression analyses. All characteristics were included in a multivariate Cox regression model. Analyses were performed using SPSS version 26.0  and R studio (version 1.1.383) with R CRAN . All statistical tests were two-sided, and P < 0.05 was considered statistically significant.Clinical characteristics were compared between the surgery and non-surgery groups using χWe conducted sensitivity analyses to examine the robustness of our results. We first used a multivariate Cox proportional hazards regression model to assess the hazard ratios (HRs) with 95% confidence intervals (CIs) for the risk factors of OS in the unadjusted data. We then carried out IPTW to adjust the differences in covariates between the surgery and the non-surgery groups. We also used the 1:1 PSM method to mimic the conditions of randomized clinical trials and reduce selection bias between the two groups , 23. FinP < 0.001; Fig. Study flow diagram was shown in Fig. P = 0.02; surgery, HR: 0.68, 95%CI: 0.51–0.91, P = 0.01; target therapy or immunotherapy, HR: 0.70, 95%CI: 0.53–0.93, P = 0.01, Tables P = 0.03, Table P = 0.02; surgery, HR: 0.58, 95%CI: 0.40–0.84, P = 0.004, Table All characteristics were included in the multivariate Cox regression model Table . In the P = 0.01), PSM-adjusted data , and IPTW-adjusted data . In lung adenocarcinoma or squamous carcinoma BMs with KPS ≤ 70, neurosurgical resection was associated with a good prognosis after PSM or IPTW adjustment .In the Kaplan–Meier survival plot Fig. , patientThe results of this multicenter, retrospective cohort study showed that neurosurgical resection improved OS and was associated with a significantly better prognosis in patients with lung cancer BMs and poor KPS. Differences in baseline characteristics between the two groups were balanced and the association between neurosurgical resection and improved OS remained evident after IPTW or PSM adjustment.As most of BMs are located in the cerebral cortex, up to 40% of BMs present with focal neurological deficits, while increased intracranial pressure from mass effect and vasogenic edema is also common. This often leads to altered mental status or impaired cognition, which makes KPS worse . NeurosuEuropean Association of Neuro-Oncology guidelines recommend that surgical resection can be performed in patients with BMs and a KPS ≥ 60 and controlled systemic disease , while tDespite the positive effect of surgical resection on OS in patients with lung cancer BM and a poor KPS in the current study, the response to surgical resection may differ among individuals, and surgical candidates should thus be selected carefully. Potential prognosis factors should be considered, including age, number of BMs, neurocognitive function, status of the primary cancer and systemic disease, genetic testing, and routine preoperative examination .This study had several strengths. First, to the best of our knowledge, this was the first multicenter, retrospective study to investigate the survival benefit of neurosurgical resection in patients with lung cancer BMs and poor KPS, thus providing a clinical reference. Second, we performed IPTW and PSM to adjust for various potentially confounding factors, and showed that the baseline characteristics were balanced after adjustment. Third, we performed multivariate Cox regression models before and after IPTW and PSM adjustment, to verify the results. All the results consistently indicated that neurosurgical resection was an independent protective factor for OS, suggesting that the study results were robust.However, the study also had some limitations. First, it was a non-randomized study and potential flaws might have remained, despite IPTW and PSM. Second, after PSM, nearly half of the patients were unmatchable and excluded, probably due to differences in covariates between the two groups.Neurosurgical resection improved OS and was associated with a significantly better prognosis in patients with lung cancer BMs and poor KPS. These findings suggest that a poor KPS should not be a contraindication for surgical resection in patients with lung cancer BMs.Supplementary file1 (DOCX 2437 KB)Below is the link to the electronic supplementary material.


Denosumab is a RANK-L inhibitor used off-label as a treatment for a variety of pediatric bone disorders, including aneurysmal bone cysts (ABC). Rebound hypercalcemia is a known side effect after denosumab therapy and is more commonly reported in pediatric patients. Although there are no established treatment guidelines, denosumab-induced rebound hypercalcemia is usually managed with a combination of intravenous fluids, diuretics, corticosteroids, denosumab, and/or bisphosphonates. We present the case of a 10-year-old female patient with history of a right sacral ABC treated with denosumab who presented with recurrent episodes of rebound hypercalcemia beginning 3 months after denosumab cessation. After the third hospitalization for hypercalcemia, which was treated with zoledronic acid, normocalcemia was achieved. This case demonstrates an increasingly recognized side effect of denosumab therapy that occurs mainly in skeletally immature patients and presents a possible approach to initial therapy of rebound hypercalcemia with a long-acting bisphosphonate. Rebound hypercalcemia following treatment with denosumab is a known side effect that is more frequently reported in skeletally immature patients . CurrentA 10-year-old female individual with no prior medical history initially presented at age 8 with progressive low back pain, right foot drop, and refusal to bear weight on the right lower extremity. Magnetic resonance imaging showed a multiloculated cystic lesion within the right sacrum with encroachment into the S1 neural foramina. She underwent urgent vascular embolization of the medial sacral artery, curettage, and S1 decompression, with intraoperative pathology confirming the diagnosis of an ABC. She re-developed symptoms several months later and required ABC aspiration with bone cement placement. The multidisciplinary tumor board recommended medical treatment with denosumab due to failure of surgical management and ongoing symptoms. Prior to denosumab initiation, laboratory tests were notable for hypophosphatemia , hyperchloremia , and low 25-hydroxy vitamin D . Her complete blood count, magnesium, and the remainder of the complete metabolic panel were unremarkable.Denosumab therapy was initiated, with 60 mg per dose for 22 total doses based on a previously described protocol: weekly for 4 doses, every 4 weeks for 12 doses, and every 8 weeks for 6 doses [Monitoring labs collected 108 days after the last denosumab dose showed hypercalcemia , hyperphosphatemia , and low 25-hydroxy vitamin D . That same day, she developed progressive nausea, vomiting, abdominal pain, diarrhea, and fatigue. She presented to the emergency room on day 3 of symptoms.Her initial exam was notable for elevated blood pressure (122/75 mmHg), tachycardia (heart rate 110 bpm), and dry mucous membranes. Blood tests demonstrated serum calcium of 15.3 mg/dL , albumin 4.3 g/dL , ionized calcium 1.65 mmol/L , parathyroid hormone related protein of <2.0 pmol/L , and appropriately suppressed intact parathyroid hormone . Creatinine was within reference range  but elevated from her baseline ranging from 0.3-0.59 mg/dL. Urine calcium was appropriately detectable at 16.3 mg/dL with a urine calcium to urine creatinine ratio of 1.63 mg/mg . Thyroid function tests and morning cortisol were within normal limits.Initial management included intravenous 0.9% saline with 5% dextrose at a rate of 3.55 to 4.75 mL/kg/h and furosemide. Furosemide was held intermittently to prevent exacerbation of acute kidney injury, with creatinine peaking at 1.16 mg/dL  on hospital day 2. A renal ultrasound was obtained due to concern for nephrocalcinosis, which was unremarkable. Due to persistence of hypercalcemia, pamidronate was initiated on hospital day 1. The recommended pamidronate dose for severe hypercalcemia is 1.5-2 mg/kg. In collaboration with our pediatric pharmacist, we decided on an initial dose of 1 mg/kg due to her relative kidney injury.Ionized calcium gradually normalized by hospital day 4. Two episodes of hypocalcemia occurred during hospitalization, both of which normalized without intervention. She was discharged on hospital day 7 with cholecalciferol 1000 IU daily.Five days after discharge (121 days after denosumab cessation), she was readmitted for hypercalcemia  associated with abdominal pain and emesis. She was treated with intravenous lactated ringers with 5% dextrose at a rate of 4.80 mL/kg/h, furosemide, a second pamidronate 1 mg/kg infusion, and methylprednisolone 1 mg/kg twice daily for 5 days. Two days after discharge from the second hospitalization (135 days after denosumab cessation), she was admitted for asymptomatic hypercalcemia detected on outpatient laboratory monitoring: ionized calcium 1.77 mmol/L . She was treated with one dose of zoledronic acid 0.03 mg/kg on hospital day 1, methylprednisolone 1 mg/kg twice daily for 3 days (discontinued early due to hypertension), and intravenous 0.9% saline and 0.45% saline with 20 mEq potassium chloride at a rate of 3.61 to 4.77 mL/kg/h. Calcium levels normalized by hospital day 2 and she was discharged on hospital day 6. Ionized calcium trends during the 3 hospitalizations are demonstrated graphically in Denosumab is a human monoclonal antibody that binds and sequesters RANK-L, thereby preventing the RANK-L/RANK interaction that leads to osteoclast differentiation and activity. Unlike bisphosphonates which bind to hydroxyapatite in the bone matrix for many years, denosumab does not bind to the bone matrix and therefore its effects are reversible. Rebound hypercalcemia is a known adverse effect of denosumab therapy. Although the exact mechanism is not clear, it is hypothesized to be caused by rapid bone resorption following recovery of osteoclast activity. Increased proportions of osteoclast precursors have been detected in peripheral blood mononuclear cells following treatment with denosumab . DenosumThe pharmacokinetics and pharmacodynamics of denosumab have been studied in adults but not children . ReboundABCs are benign bone lesions that occur most frequently in the first 2 decades of life. Although benign, they often cause a significant amount of local destruction and clinical symptoms, including pain and neurologic effects. Treatment options for ABCs include surgical resection, intralesional curettage, sclerotherapy, radiotherapy, and selective arterial embolization . Axial AAlthough no established treatment recommendations exist, the management of denosumab-induced rebound hypercalcemia typically involves a combination of intravenous hydration, diuretics, calcitonin, corticosteroids, bisphosphonates, and/or repeat dosing of denosumab. We elected to use a combination of hydration, furosemide, corticosteroids, pamidronate, and zoledronic acid. Calcitonin was considered but not pursued due to risk of tachyphylaxis and unavailability in the hospital formulary at the time of this patient's admission. Similarly, denosumab was avoided due to concern for inciting further episodes of rebound hypercalcemia. Because steroids inhibit enteral absorption of calcium, we administered a course of steroids after her second admission to mitigate further exacerbation of hypercalcemia. The patient's first 2 episodes were managed with pamidronate, a bisphosphonate with a half-life of approximately 28 hours. Hypercalcemia recurred 11 days  and 14 days  after pamidronate infusions, respectively. Her third episode was treated with zoledronic acid, a bisphosphonate with a half-life of 146 hours. Because long-lasting normocalcemia was achieved after administration of zoledronic acid, we hypothesize that initial management with a bisphosphonate with a longer half-life may have been more effective in preventing subsequent episodes of hypercalcemia. Alternatively, the rebound resorptive effect post denosumab may have diminished by the end of her third hospitalization, regardless of the type of bisphosphonate that was used. Another interesting approach is treatment targeting prevention of anticipated rebound hypercalcemia. Seale et al reported a strategy involving alternating doses of denosumab and zoledronic acid every 3 months in a young boy with osteogenesis imperfecta type IV after an episode of denosumab-induced rebound hypercalcemia [There are currently no guidelines regarding the timing or frequency of laboratory monitoring during or following denosumab therapy. Hypercalcemia has been reported to occur from less than 1 month to 7 months following denosumab cessation . More reBecause denosumab is a promising agent in the treatment of pediatric ABCs, it is important to better understand the risk factors, prevention, and best management for its adverse effect of rebound hypercalcemia. Early management with longer lasting bisphosphonates such as zoledronic acid may be beneficial in preventing repeat episodes of hypercalcemia. Future research should aim to further examine the role of zoledronic acid as a first-line treatment for rebound hypercalcemia after denosumab cessation.Rebound hypercalcemia is a known side effect of denosumab therapy that is more frequently reported in skeletally immature patients and typically develops 3 to 5 months after denosumab discontinuation.Because denosumab-induced rebound hypercalcemia often presents with acute symptoms of hypercalcemia before it is detected on routine laboratory monitoring, it is important to educate patients and families on the symptoms of hypercalcemia so they can promptly seek treatment.Long-acting bisphosphonates, such as zoledronic acid, should be considered in the early management of denosumab-induced rebound hypercalcemia, as they may prevent subsequent episodes of rebound hypercalcemia.