Objective To evaluate the relationship between preoperative COVID-19 infection and major postoperative pulmonary complications (PPC) risk after major elective surgeries during the Omicron wave.Design A multicentre, prospective, observational cohort study.Setting Four tertiary medical centres in Beijing, China.Participants All adult patients who underwent major elective surgeries under general anaesthesia from 30 December 2022 to 18 May 2023 were screened for eligibility. A total of 3211 patients were included.Primary and secondary measures The primary outcome was 30-day major PPC, defined as pneumonia, acute respiratory distress syndrome or unexpected postoperative ventilation. The secondary outcomes included length of hospital stay (LOS), reoperation and mortality.Results Major PPC occurred in 3.5% of patients with preoperative COVID-19 and 3.3% of those without. Inverse probability of treatment weighting-adjusted analysis showed no significant association between preoperative COVID-19 within 12 weeks and PPC risk (adjusted OR, 0.89; 95% CI 0.69 to 1.13). However, multivariable analysis revealed that COVID-19 infection within 3 weeks was independently associated with an increased PPC risk (OR, 3.44; 95% CI 1.37 to 8.68). Cardiothoracic surgery (OR, 12.47; 95% CI 8.11 to 19.17) and longer duration of surgery (OR, 1.24 per hour; 95% CI 1.13 to 1.37) were significant risk factors. In the cardiothoracic subgroup, PPC risk was significantly elevated within 7 weeks of infection. No significant differences were observed in LOS, reoperation rates or mortality between patients with and without preoperative COVID-19 infection.Conclusions Preoperative COVID-19 infection within 12 weeks was not associated with an increased overall risk of major PPC during the Omicron wave. Although very short infection-to-surgery intervals and cardiothoracic surgery showed exploratory signals of higher risk, these findings should be interpreted cautiously and support an individualised approach to perioperative risk assessment.Trial registration number ChiCTR2200067250.

Background:Effective postoperative pain management is vital for the recovery, mobility, and quality of life of patients undergoing anterior cruciate ligament reconstruction (ACLR) surgery. Our retrospective analysis aims to identify factors affecting postoperative breakthrough pain after ACLR, with a focus on analgesic modalities. Methods:This retrospective study included 848 patients who underwent ACLR surgery at Peking University Third Hospital from January 1, 2019, to September 30, 2022. After applying exclusion criteria, patients were categorized into four groups: general anesthesia with femoral nerve block (Group G + F, n = 156), general anesthesia alone (Group G, n = 129), spinal anesthesia with femoral nerve block (Group S + F, n = 314), and spinal anesthesia alone (Group S, n = 249). The collected data included demographic details, analgesic methods, and the timing of the first pain relief request. Statistical analysis was performed using the chi-square test and multivariable logistic regression to evaluate intergroup differences and the effect of covariates. Results:A significant difference was found in the timing of the first analgesic request among the groups (p < 0.001). Group G and Group S requested their initial postoperative analgesics sooner than Groups G + F and S + F, respectively (p < 0.001). Multivariable analysis showed that gender was associated with postoperative breakthrough pain (odds ratio [OR] 0.502; 95% confidence interval [CI] 0.307 to 0.821). Postoperative routine use of non-steroidal anti-inflammatory drugs (NSAIDs) also reduced breakthrough pain incidence (OR 0.286; 95% CI 0.182 to 0.451). Breakthrough pain at 12 and 24 hours post-surgery was associated with prolonged hospital stays (p = 0.001 and 0.006, respectively). Conclusion:The administration of a single-shot femoral nerve block (FNB) significantly delayed the first request for analgesia after ACLR, without leading to prolonged hospitalization. Factors associated with postoperative breakthrough pain during hospitalization included gender, regular NSAID use, and analgesic modality selection.

Cellular membranes serve as selective barriers, and membrane permeability is crucial for drug pharmacokinetics. While in vitro and in vivo methods exist, predicting and designing membrane permeability remains challenging. We developed a thermodynamics-based deep learning framework to analyze the structure-permeability relationship, based on the concept that interactions between membrane lipids and small molecules influence permeability. We determined the membrane penetration thermodynamics of 8,239 compounds using coarse-grained molecular dynamics simulations and created interpretable graph neural network models to predict and design drug membrane permeability. As a proof-of-concept, we designed a novel nasal-administered melatonin analog, MT-A2, optimized for permeability. Compared with melatonin, MT-A2 showed superior nasal absorption, prolonged brain retention, and enhanced sleep efficacy. Our results provide a promising approach for predicting and designing membrane permeability, aiding in the development of drugs with better pharmacokinetics.

Postoperative delirium (POD) is common in elderly surgical patients and has significant implications for patient recovery. This study aimed to investigate the association between cerebral small vessel disease (CSVD) imaging markers and the occurrence of POD in elderly surgical patients. Eighty-two elderly patients who had undergone surgery were retrospectively examined. The case group comprised 41 patients who developed POD, while the control group consisted of 41 who did not. The groups were matched 1:1 with respect to age, sex, surgery type, and operation duration. Preoperative brain magnetic resonance imaging was analyzed for CSVD markers, including white matter hyperintensities (WMHs), perivascular spaces (PVSs), lacunar infarcts, and global cortical atrophy. Logistic regression was used to assess the relationship between CSVD markers and POD. WMHs, PVSs, and lacunar infarcts significantly differed between the groups. Univariate analysis showed no significant effect for individual CSVD markers on POD. However, deep WMHs and white matter PVSs emerged as the most important independent predictors after stepwise multivariate regression. The final model showed that deep WMHs (odds ratio, 2.34; 95

Background:Postoperative delirium (POD) is a common complication after cardiac surgery, but the role of preoperative gut microbiota, especially the mycobiome, in POD susceptibility remains unclear. This study investigated the association between preoperative gut bacterome and mycobiome profiles and POD in elderly patients undergoing minimally invasive direct coronary artery bypass grafting (MIDCABG). Methods:Preoperative fecal samples from 31 elderly patients scheduled for MIDCABG were analyzed via 16S rRNA and ITS high-throughput sequencing. POD was assessed twice daily using the 3D-CAM scale. Patients were divided into delirium (M, n = 7) and control (C, n = 24) groups. Microbial diversity, composition, and bacteria-fungi correlations were systematically evaluated. Results:POD incidence was 22.58%. No baseline differences were observed between groups. Group M exhibited significantly lower gut microbiome health index and higher microbial dysbiosis index at both bacterome and mycobiome levels. Alpha/beta diversity did not differ significantly. LEfSe revealed bacterial enrichment of Clostridium and Intestinibacter in Group M, and Bifidobacterium in Group C. Fungally, Aspergillus and Basidiomycota predominated in Group M, while Saccharomyces and Ascomycota were enriched in Group C. candida showed the highest abundance in Group M and was a key node in the fungal co-occurrence network. Correlation analysis revealed positive associations between bacterial genera (Klebsiella, Enterococcus, Roseburia) and fungal taxa (Wickerhamomyces, Pichia, Geotrichum), suggesting ecological interactions potentially linked to POD. Conclusion:Preoperative dysbiosis of gut bacterome and mycobiome is associated with POD in elderly MIDCABG patients. Several bacterial and fungal taxa, including Clostridium, Intestinibacter, Aspergillus, Saccharomyces, and Candida, were identified as candidate microbial biomarkers associated with POD and may contribute to future microbiota-based risk stratification strategies.

BackgroundAlthough altered grip strength is a recognized predictor of various central nervous system disorders, large-scale clinical evidence linking it to delirium and postoperative delirium (POD) remains scarce. Using data from the UK Biobank (UKB), this study investigated the association between baseline grip strength and the future risk of incident delirium.MethodsWe analyzed 452,711 UKB participants without delirium at baseline. Grip strength was measured at study entry. Incident delirium was identified from hospital records using ICD-10 code F05. POD was defined by the occurrence of surgery before delirium diagnosis, using postoperative windows of 3 and 7 days. Accelerated failure time (AFT) were used in the primary analyses to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). The proportional hazards assumption was assessed using Schoenfeld residual tests. Because sex modified the associations of interest, sex-stratified analyses were performed. Restricted cubic spline analyses and Kaplan-Meier curves were used to characterize dose-response patterns and cumulative incidence.ResultsOver a median follow-up of 14.9 years, 7695 incident delirium events were identified. Lower baseline grip strength was associated with higher risks of incident delirium and POD. Sex modified these associations, particularly for incident delirium and POD defined within 3 days after surgery. In both males and females, restricted cubic spline analyses supported inverse associations between grip strength and the risks of incident delirium, POD within 3 days, and POD within 7 days. Kaplan-Meier curves showed higher cumulative incidence across decreasing grip strength tertiles. In fully adjusted models, lower grip strength remained associated with higher risks across outcomes in both sexes. The lowest grip strength tertile consistently showed the highest risk. In females, however, the intermediate tertile was associated with incident delirium but not with POD.ConclusionLower baseline grip strength was independently associated with higher risks of incident delirium and POD, with modest sex-related differences in the strength of association. Grip strength measurement may offer a simple, noninvasive means of identifying individuals at increased risk of delirium-related outcomes.

IntroductionPropofol may induce emotional impairment like euphoria and elation. Previous studies have demonstrated that emotional impairment can injure social cognition like emotion recognition and decision-making abilities. Therefore, this study is designed to investigate the effects of propofol anesthesia on facial emotion recognition (FER) and delay discounting behavior.MethodPatients underwent diagnostic gastrointestinal endoscopy (GI) with propofol anesthesia in this prospective cohort observational study. Prior to and following the procedure (approximately 30 min afterwards), patients were asked to select the word that best describes the presented facial photographs displaying happiness, anger, and neutral expressions. Additionally, Monetary Choice Questionnaire-9 was used to assess delay discounting.ResultsWithin a cohort of 87 patients, 11 patients (12.6%) met the criterion of FER deficit post-GI. The FER of anger exhibited significant differences between pre- and post-GI, considering both the correct (52.6%) and incorrect (24.3%) recognition. There was a positive identification bias for FER after propofol anesthesia: mistaking anger (p = 0.02) or neutral (p = 0.01) expression for happiness. Procedures in the morning and the absence of insomnia were associated with the decreased FER score of anger post-GI. The results did not indicate any impairment of propofol anesthesia on FER of happiness or delay discounting behavior.ConclusionThe study demonstrates that propofol anesthesia during GI endoscopy selectively impairs the recognition of anger facial expressions while leaving the recognition of happiness and delay discounting unaffected at a short-term postoperative observation. Additionally, the recognition of anger and neutral facial expressions exhibited a tendency towards a positive bias.Clinical trial registrationhttps://www.chictr.org.cn/showproj.html?proj=199458, identifier ChiCTR2300073132.

General anesthetics profoundly alter brain function and consciousness, yet the mechanisms underlying these effects remain incompletely understood. Although traditional studies have primarily focused on neuronal targets, accumulating evidence suggests that microglia dynamically respond to anesthetic exposure and may participate in anesthesia-associated neurophysiological changes. Beyond their established immune functions, microglia are increasingly implicated in synaptic remodeling, metabolic regulation, neuronal activity surveillance, and neuron-glia communication. Recent studies indicate that different classes of anesthetic agents modulate microglial activity through diverse and context-dependent mechanisms involving inflammatory signaling, purinergic pathways, calcium dynamics, mitochondrial metabolism, and neural circuit interactions. These responses are associated with postoperative neurocognitive disorders, altered synaptic plasticity, and anesthesia-related changes in brain states. In this review, we summarize current evidence regarding the effects of volatile anesthetics, intravenous anesthetics, and analgesics on microglial function and discuss the molecular, functional, and circuit-level mechanisms underlying anesthesia-associated neuron-microglia interactions. We further highlight the dynamic and heterogeneous nature of microglial responses during anesthesia and discuss current limitations in the field, including the lack of temporally resolved and cell-specific approaches. Understanding these processes may provide insights into anesthesia-associated neurocognitive dysfunction and support the development of neuroimmune-targeted strategies in anesthesiology.

Painful diabetic neuropathy (PDN) is frequently accompanied by anxiety, yet the neural circuit mechanisms associated with nociceptive hypersensitivity to affective dysfunction remain unclear. Here we combined brain-wide c-Fos mapping, viral tracing, in vivo fiber photometry, whole-cell recordings, and projection-specific opto/chemogenetics in a mouse model of streptozotocin-induced PDN to define a thalamo-cortex circuit for pain-anxiety comorbidity. Within the posterior insular cortex (pIC), CaMKIIα⁺ excitatory neurons were recruited in PDN mice with comorbid anxiety, exhibiting heightened neuronal excitability and enhanced excitatory synaptic input. Bidirectional chemogenetic modulation of pICCaMKIIα neurons oppositely regulated anxiety-like behavior and mechanical hypersensitivity without locomotor or glycemic confounds. Circuit mapping identified a monosynaptic excitatory projection from the paraventricular thalamus (PVT) to the pIC; PVTCaMKIIα neurons were hyperactive in PDN, and projection-defined manipulation of the PVTCaMKIIα to pICCaMKIIα pathway bidirectionally controlled the behavioral phenotypes. Cacna1c was upregulated in the PVT of PDN mice; its knockdown in pIC-projecting PVTCaMKIIα neurons attenuated both pain and anxiety-like behaviors. Together, these results delineate a thalamo-cortex excitatory pathway that couples nociceptive and affective dimensions of PDN and suggest that selective targeting of the PVTCaMKIIα to pICCaMKIIα circuit may provide coordinated relief of pain and anxiety-related symptoms.

Spinal anesthesia-induced hypotension (SAIH) is a common complication during cesarean delivery and is closely associated with maternal intravascular volume status. However, reliable real-time assessment of volume changes remains challenging. This study aimed to evaluate whether transthoracic echocardiography (TTE)-derived ventricular volume parameters could dynamically reflect peri-anesthetic volume changes and predict SAIH. A total of 100 healthy singleton parturients scheduled for elective cesarean delivery under spinal anesthesia were prospectively enrolled. Left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) were measured by transthoracic echocardiography using the biplane Simpson method at three predefined time points: after admission (T1), after crystalloid fluid preloading (T2), and after stabilization of the sensory block (T3). Systolic blood pressure (SBP), mean arterial pressure (MAP), and heart rate (HR) were recorded simultaneously. SAIH was defined as an SBP < 90 mmHg or a decrease of > 30

Deep anesthesia is associated with delayed recovery, postoperative cognitive dysfunction, and increased long-term mortality. In clinical practice, improper control of anesthesia depth can lead to intraoperative awareness or excessive suppression, thereby elevating the risk of postoperative neurological complications. Therefore, accurate monitoring and prediction of anesthesia depth are of great clinical importance. Currently, the bispectral index (BIS) is the most widely used clinical tool for assessing anesthesia depth by analyzing electroencephalogram (EEG) signals. However, existing BIS monitoring models can only provide real-time assessments and cannot effectively predict the trend of anesthesia depth. To address this limitation, this study proposes AnesNet, a deep learning–based model designed to provide early warnings of anesthesia depth 5, 10, and 15 min in advance. Considering diverse clinical needs, AnesNet supports both regression and classification prediction paradigms, enabling continuous depth forecasting and risk-oriented early warning of clinically unfavorable anesthesia states. A total of 2,803 surgical patients were included in this study. Results show that AnesNet achieved an AUROC of 0.895 in classification tasks and a MAE of 4.90 in regression tasks. Furthermore, feature ablation experiments demonstrated the model’s interpretability, enabling quantification of the contribution of individual physiological parameters to the prediction outcomes.

BACKGROUND:Surgery/anesthesia is major physiological stressors related to biological aging, while physical activity (PA) is known to have anti-aging effects. However, the relationship between PA and surgery-associated biological aging remains unclear. Therefore, we aimed to investigate the association between surgical history and biological aging and to examine whether PA modifies this relationship. METHODS:This cross-sectional study analyzed data from the UK Biobank, including 126,668 participants aged ≥60 years with complete surgical histories, PhenoAge biomarker data, and relevant covariates. Three analyses assessed associations between biological aging and number of major surgeries, shortest interval between adjacent surgeries, and time since most recent surgery. PA was assessed using questionnaire-derived MET-min/week, and PA levels were categorized as high (≥600) or low (<600 MET-min/week). Biological aging was measured using PhenoAge acceleration (PhenoAgeAccel), calculated from chronological age and nine clinical biomarkers (higher values indicate accelerated aging). Multivariable linear regression models adjusted for demographic characteristics, lifestyle factors, and comorbidities. RESULTS:Higher cumulative surgical exposure was significantly associated with PhenoAgeAccel. Compared to those with no surgeries, participants with ≥4 surgeries had a 0.14 to 0.39-year PhenoAgeAccel. Shorter surgical intervals (≤1 year) were associated with a 0.11-year PhenoAgeAccel compared to intervals >5 years. More recent surgery (≤1 year) was associated with a 0.35-year PhenoAgeAccel. High PA attenuated these associations. In high-PA individuals, statistically significant aging was observed only with ≥5 surgeries (vs ≥4 in low-PA) and within one year of recent surgery. No significant acceleration was observed in high-PA individuals with short surgical intervals. CONCLUSION:Number of surgeries, short surgical intervals, and recent surgery are associated with accelerated biological aging, which indicates that optimizing surgical timing may represent a potential strategy to mitigate the biological burden associated with repeated surgical exposure. Furthermore, the level of PA is negatively correlated with surgery-related aging.

BACKGROUND:Delirium is an acute brain dysfunction syndrome with high morbidity and mortality, however its mechanisms remain poorly understood. Although multiple risk factors have been identified, directly targeting them is challenging. Proteins may provide new insights into this challenge. METHODS:Using data from the UK Biobank, we explored associations between 18 established delirium risk factors and incident delirium using Cox proportional hazards models. Plasma proteomic profiling was conducted to identify proteins associated with delirium. Identified proteins underwent enrichment and mediation analyses to evaluate their potential involvement in the associations between risk factors and delirium. RESULTS:Over a median 13.5-year follow-up, 701 participants developed delirium. Among 2915 plasma proteins, 78 were significantly associated with delirium after Bonferroni correction, including Neurofilament light polypeptide (NEFL), Glial fibrillary acidic protein, WAP four-disulfide core domain protein 2 (WFDC2), Urokinase plasminogen activator surface receptor, and Growth/differentiation factor 15 (GDF15). Enrichment analysis suggested that these proteins were predominantly involved in immune, inflammatory, and metabolic processes. Mediation analysis indicated that some proteins may statistically mediate the associations between clinical risk factors and incident delirium; for example, NEFL (11.7%) and EDA2R (10.4%) for age, PLAUR (47.2%) and Mesothelin (MSLN; 40.1%) for smoking, GDF15 (40.6%) and C7 (16.3%) for diabetes, WFDC2 (13.4%) and PLAUR (13.1%) for frailty. Overall, 24 proteins statistically mediated the associations between at least 9 risk factors and delirium, with GDF15, PLAUR, Amphiregulin, Hepatitis A virus cellular receptor, Occludin, and, showing relatively higher statistical estimates. CONCLUSIONS:This large-scale study identifies plasma proteins that may help explain some of the observed associations between clinical risk factors and incident delirium, generating hypotheses about underlying mechanisms and potential avenues for further research.

ObjectiveTo compare the efficacy and safety of pharmaceutical interventions to prevent etomidate-induced myoclonus (EIM), providing the optimal intervention for clinical practice.MethodsPubMed, Embase, the Cochrane Central Register of Controlled Trials, ClinicalTrials.gov, Chinese National Knowledge Infrastructure, WanFang database, and SinoMed database were searched from the inception to sixth May 2024. We included randomized controlled trials (RCTs) comparing intravenous pharmaceutical interventions to prevent EIM with placebo, no intervention, or another pharmaceutical intervention.ResultsForty-eight RCTs involving 4,768 participants randomly assigned to 20 intravenous pharmaceutical interventions and normal saline were included. Granisetron (odds ratio [OR]: 0.01, 95% confidence interval [CI]: 0.00 to 0.06; one study, moderate certainty) and oxycodone (OR: 0.01, 95% CI: 0.00 to 0.05; three studies, low certainty) was found to be the most effective intervention in reducing the risk of EIM and ranked highest in terms of surface under the cumulative ranking values (94.4% and 89.7% probability), followed by sufentanil (76.5% probability) and remifentanil (74.8% probability). Further subgroup analysis of EIM at mild, moderate-to-severe levels highlighted granisetron and oxycodone as the favorable interventions for reducing EIM. For safety outcomes, the synthesized results indicated that opioids were associated with a higher risk of adverse events (AEs), while no severe AEs were observed.ConclusionModerate-to-low certainty evidence indicated that granisetron and oxycodone may represent the optimal intervention for reducing the risk of overall and moderate-to-severe EIM with a reasonable safety profile, providing the potential interventions for clinical practice.Systematic Review Registrationhttps://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=291275.

OBJECTIVES:To improve the medical treatment process, the concept of comfortable medical care has been proposed. This concept has been fully implemented in developed countries, but there are still many restrictions on its promotion and implementation in developing countries. This study investigates public preferences about comfortable medical care and identifies the factors influencing these in China. DESIGN:A cross-sectional survey using an online questionnaire. We used multivariate logistic regression analysis to identify the factors influencing preferences for comfortable medical care and developed a prediction model of preferences for four types of this care among the general population. SETTING:Peking University Third Hospital, China. PARTICIPANTS:Members of the general public with no medical or biomedical background. PRIMARY AND SECONDARY OUTCOME MEASURES:The measures included demographic information and attitudes towards four types of comfortable medical services. The primary outcome was attitude towards general anaesthesia. RESULTS:Overall, 5330 people participated in the survey. Nearly 80% would choose painless gastroenteroscopy, labour analgesia and postoperative analgesia. Interestingly, just 43.9% preferred general anaesthesia over regional anaesthesia. The impact of general anaesthesia on learning and memory (69.8%) was the most worrying issue. Younger people with higher education levels and incomes, and living in cities, preferred painless medical services. In the prediction model for general anaesthesia, the areas under the receiver operating characteristic curves were 0.790 (95% CI 0.776 to 0.804) in the development group and 0.777 (95% CI 0.754 to 0.799) in the internal validation group. CONCLUSION:The Chinese public has a good level of acceptance of comfortable medical care, although there are still many obstacles to its popularisation and promotion. Our prediction model could be used to screen the population for targeted popularisation work to expand the implementation of this approach.

Background:Postoperative delirium (POD) is a common central nervous system complication in older adult surgical patients. At present, the mechanism for POD is still unclear. Lipocalin-2 (LCN2) may have an impact on cognitive function, but the relationship between LCN2 and POD has remained unclear. Therefore, we sought to investigate the relationship between the levels of LCN2 in plasma and cerebrospinal fluid (CSF) and the occurrence of POD in older adults undergoing hip fracture surgery. Methods:We conducted a prospective observational cohort study involving 186 older adults (≥65 years old) who underwent hip fracture surgery under spinal anesthesia. CSF and blood samples were collected. The levels of LCN2, interleukin-6 (IL-6), and interleukin-1 (IL-1) were measured using an enzyme-linked immunosorbent assay (ELISA). We used the 3-min diagnostic interview to evaluate delirium defined by the Confusion Assessment Method (3D-CAM), to screen for POD, and the Memorial Delirium Assessment Scale (MDAS) to evaluate the severity of delirium. Multivariable logistic regression was applied to identify independent predictive factors for POD. The relationship between CSF LCN2 levels and POD risk was assessed through receiver operating characteristic (ROC) curve analysis. Correlation analysis was used to investigate the association between CSF LCN2 and MDAS scores as well as IL-6. Results:Among the 186 patients ultimately included, 29 (15.6%) developed POD. Their preoperative CSF LCN2 level was significantly higher than that of those without POD (p = 0.001). The multivariable logistic regression analysis revealed that an elevated preoperative CSF LCN2 level [odds ratio (OR) 2.546, 95% confidence interval (CI) 1.345-4.822; p = 0.004] was an independent predictor of POD. Moreover, among POD group patients, preoperative CSF LCN2 levels were positively correlated with the MDAS scores (r = 0.688, p < 0.001) and CSF IL-6 levels (r = 0.379, p = 0.043). ROC analysis of preoperative CSF LCN2 showed an area under the curve of 0.713 (95% CI 0.615-0.810) with a specificity of 75.0%, and sensitivity of 58.6%. Conclusion:Elevated preoperative CSF LCN2 levels are associated with an increased risk and severity of POD in older adults undergoing hip fracture surgery. Clinical trial registration:https://www.chictr.org.cn/, ChiCTR2200061407.

BACKGROUND:Malignant hyperthermia (MH) is a life-threatening pharmacogenetic disorder triggered by certain anesthetics, characterized by muscle rigidity, elevated body temperature, and hypermetabolic crisis. This condition is primarily associated with genetic mutations in ryanodine receptor 1 ( RYR1 ), which encodes the pivotal calcium release channel in the sarcoplasmic reticulum of skeletal muscle. While numerous hotspot mutations in RYR1 have been identified, the functional impact of nonhotspot mutations on channel activity related to MH remains insufficiently investigated. In this study, we identified a known pathogenic mutation (p.Arg2508His) and a novel variant (p.Asp2730Tyr), both located outside the conventional MH hotspots, in 2 patients with clinical suspicion of MH. Our objective was to investigate the functional implications of the p.Asp2730Tyr mutation in RYR1 on calcium release dynamics related to MH. METHODS:We engineered a recombinant wild-type (WT) plasmid (pcDNA3.1-3Myc-His-RYR1-WT) to express the full-length mouse skeletal muscle RYR1 using seamless multi-fragment cloning techniques. Two RYR1 mutations, p.Arg2508His (used as positive control) and p.Asp2730Tyr, were separately introduced into the WT plasmid, generating 2 mutant constructs (pcDNA3.1-3Myc-His-RYR1-p.Arg2508His and pcDNA3.1-3Myc-His-RYR1-p.Asp2730Tyr). We utilized 293T cells expression system to express either the WT or mutant forms of mouse RYR1 . Fluo-4 calcium imaging was conducted to evaluate the alterations in calcium release in response to RYR1 agonists, caffeine or 4-chloro-m-cresol (4CmC), for each mutation compared to WT. RESULTS:Cells transfected with the p.Arg2508His or p.Asp2730Tyr mutation demonstrated a leftward shift in the caffeine and 4CmC concentration-response curves compared to WT, suggesting an increased channel sensitivity to caffeine and 4CmC ( P < .001). The mean ± standard error of the mean (SEM) of the EC50 values for caffeine-induced calcium release was 2.56 ± 0.04 mM in WT, which significantly decreased to 1.32 ± 0.13 mM for p.Arg2508His ( P < .001) and 1.12 ± 0.09 mM for p.Asp2730Tyr ( P < .001). For 4CmC, the EC50 values were 43.2 ± 1.90 μM in WT, 17.2 ± 0.76 μM for p.Arg2508His ( P < .001), and 21.8 ± 1.04 μM for p.Asp2730Tyr ( P < .001), indicating enhanced calcium release in both mutations. CONCLUSIONS:The p.Asp2730Tyr mutation, situated beyond the established RYR1 hotspot regions, significantly alters calcium release dynamics related to MH. A comprehensive investigation into the structural conformations, functional assays, and in vivo mechanisms associated with this mutation could yield a more profound understanding of the molecular underpinnings of MH pathogenesis.