Correlative montage parallel array cryo-tomography for in situ structural cell biology.

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here we present robust tools for montage parallel array cryo-tomography (MPACT) tailored for vitrified specimens. The combination of correlative cryo-fluorescence microscopy, focused-ion-beam milling, substrate micropatterning, and MPACT supports studies that contextually define the three-dimensional architecture of cells. To further extend the flexibility of MPACT, tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

A neutralizing antibody target in early HIV-1 infection was recapitulated in rhesus macaques immunized with the transmitted/founder envelope sequence.

Transmitted/founder (T/F) HIV-1 envelope proteins (Envs) from infected individuals that developed neutralization breadth are likely to possess inherent features desirable for vaccine immunogen design. To explore this premise, we conducted an immunization study in rhesus macaques (RM) using T/F Env sequences from two human subjects, one of whom developed potent and broad neutralizing antibodies (Z1800M) while the other developed little to no neutralizing antibody responses (R66M) during HIV-1 infection. Using a DNA/MVA/protein immunization protocol, 10 RM were immunized with each T/F Env. Within each T/F Env group, the protein boosts were administered as either monomeric gp120 or stabilized trimeric gp140 protein. All vaccination regimens elicited high titers of antigen-specific IgG, and two animals that received monomeric Z1800M Env gp120 developed autologous neutralizing activity. Using early Env escape variants isolated from subject Z1800M as guides, the serum neutralizing activity of the two immunized RM was found to be dependent on the gp120 V5 region. Interestingly, the exact same residues of V5 were also targeted by a neutralizing monoclonal antibody (nmAb) isolated from the subject Z1800M early in infection. Glycan profiling and computational modeling of the Z1800M Env gp120 immunogen provided further evidence that the V5 loop is exposed in this T/F Env and was a dominant feature that drove neutralizing antibody targeting during infection and immunization. An expanded B cell clonotype was isolated from one of the neutralization-positive RM and nmAbs corresponding to this group demonstrated V5-dependent neutralization similar to both the RM serum and the human Z1800M nmAb. The results demonstrate that neutralizing antibody responses elicited by the Z1800M T/F Env in RM converged with those in the HIV-1 infected human subject, illustrating the potential of using immunogens based on this or other T/F Envs with well-defined immunogenicity as a starting point to drive breadth.

GSDMD-Mediated Cardiomyocyte Pyroptosis Promotes Myocardial I/R Injury.

[Figure: see text].

Handling Difficult Cryo-ET Samples: A Study with Primary Neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons having been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

A novel conservative system with hidden flows evolved from the simplest memristive circuit.

Over the past few decades, the research of dissipative chaotic systems has yielded many achievements in both theory and application. However, attractors in dissipative systems are easily reconstructed by the attacker, which leads to information security problems. Compared with dissipative systems, conservative ones can effectively avoid these reconstructing attacks due to the absence of attractors. Therefore, conservative systems have advantages in chaos-based applications. Currently, there are still relatively few studies on conservative systems. For this purpose, based on the simplest memristor circuit in this paper, a non-Hamiltonian 3D conservative system without equilibria is proposed. The phase volume conservatism is analyzed by calculating the divergence of the system. Furthermore, a Kolmogorov-type transformation suggests that the Hamiltonian energy is not conservative. The most prominent property in the conservative system is that it exhibits quasi-periodic 3D tori with heterogeneous coexisting and different amplitude rescaling trajectories triggered by initial values. In addition, the results of Spectral Entropy analysis and NIST test show that the system can produce pseudo-random numbers with high randomness. To the best of our knowledge, there is no 3D conservative system with such complex dynamics, especially in a memristive conservative system. Finally, the analog circuit of the system is designed and implemented to test its feasibility as well.

CorRelator: Interactive software for real-time high precision cryo-correlative light and electron microscopy.

Cryo-correlative light and electron microscopy (CLEM) is a technique that uses the spatiotemporal cues from fluorescence light microscopy (FLM) to investigate the high-resolution ultrastructure of biological samples by cryo-electron microscopy (cryo-EM). Cryo-CLEM provides advantages for identifying and distinguishing fluorescently labeled proteins, macromolecular complexes, and organelles from the cellular environment. Challenges remain on how correlation workflows and software tools are implemented on different microscope platforms to support automated cryo-EM data acquisition. Here, we present CorRelator: an open-source desktop application that bridges between cryo-FLM and real-time cryo-EM/ET automated data collection. CorRelator implements a pixel-coordinate-to-stage-position transformation for flexible, high accuracy on-the-fly and post-acquisition correlation. CorRelator can be integrated into cryo-CLEM workflows and easily adapted to standard fluorescence and transmission electron microscope (TEM) system configurations. CorRelator was benchmarked under live-cell and cryogenic conditions using several FLM and TEM instruments, demonstrating that CorRelator reliably supports real-time, automated correlative cryo-EM/ET acquisition, through a combination of software-aided and interactive alignment. CorRelator is a cross-platform software package featuring an intuitive Graphical User Interface (GUI) that guides the user through the correlation process. CorRelator source code is available at: https://github.com/wright-cemrc-projects/corr.

Neutrophil-derived advanced glycation end products-Nε-(carboxymethyl) lysine promotes RIP3-mediated myocardial necroptosis via RAGE and exacerbates myocardial ischemia/reperfusion injury.

Nε-(carboxymethyl) lysine (CML), the major member of advanced glycation end products, was widely studied in diabetic complications and aging-associated diseases. However, the impact of CML on myocardial ischemia/reperfusion injury (MI/RI) was rarely reported. In the present study, CML was increased in both patients with acute myocardial infarction (53.4 ± 7.8 vs. 28.1 ± 4.4 ng; P = 0.017), and mice underwent MI/RI (16.4 ± 1.4 vs. 10.8 ± 0.9 ng; P = 0.006). Depletion of neutrophils reduced CML (17.8 ± 1.0 vs. 9.9 ± 0.3 ng; P < 0.001), indicating neutrophils were the major cells contributing to CML formation during MI/RI. CML treatment exacerbated MI/RI by elevating myocardial injury marker (274.3 ± 18.0 vs. 477.2 ± 34.3 pg; P < 0.001), enlarging myocardial infarct size (32.9 ± 3.6 vs. 45.2 ± 3.8%; P = 0.03), increasing myocardial fibrosis (17.5 ± 1.6 vs. 29.7 ± 2.2%; P < 0.001) and impairing cardiac function (59.4 ± 2.4% vs. 46.0 ± 1.3%; P = 0.001). Further study revealed that CML increased the phosphorylation of receptor interacting protein (RIP) 3, an important initiator of necroptosis, and its downstream proteins. Receptor for advanced glycation end product (RAGE) deficiency effectively blocked RIP3 phosphorylation induced by CML and rescued CML-mediated MI/RI, indicating CML promoted RIP3-mediated necroptosis through RAGE. In addition, glyoxalase-1 overexpression could effectively attenuate MI/RI by reducing CML formation, providing a potential therapeutic target for MI/RI.-Yang, J., Zhang, F., Shi, H., Gao, Y., Dong, Z., Ma, L., Sun, X., Li, X., Chang, S., Wang, Z., Qu, Y., Li, H., Hu, K., Sun, A., Ge, J. Neutrophil-derived advanced glycation end products-Nε-(carboxymethyl) lysine promotes RIP3-mediated myocardial necroptosis via RAGE and exacerbates myocardial ischemia/reperfusion injury.

Gender-Related Differences in Clinical Characteristics and Outcomes of Premature Coronary Artery Disease: Insight from the FOCUS Registry.

INTRODUCTION: Although coronary artery disease (CAD) presentations and clinical outcomes differ by sex, little is known about premature CAD (PCAD). The present analysis aimed to evaluate the gender-related differences of PCAD in an Asian population from the FOCUS registry. METHODS: A total of 1397 Asian young patients with angiographically confirmed CAD undergoing drug-eluting stent implantation were included in this analysis and divided into two groups according to the genders. Patients were followed up for three years and clinical outcomes were compared between groups. RESULTS: Young women were older and more likely to have hypertension and diabetes than men (all p<0.001). In contrast, males with PCAD had higher BMI and higher prevalence of current smoking as well as previous vessel revascularizations (all p<0.05). Men were more likely to be manifested as total occlusive lesions (p<0.001). Regardless of the clinical characteristics, the cumulative incidences of adverse events such as major adverse cardiovascular event (MACE), cardiovascular death, and all-cause death were not significantly different at one- or three-year follow-up (all p>0.05). CONCLUSION: Despite remarkable differences in clinical characteristics between Asian males and females with PCAD, the two groups did not differ significantly in clinical outcomes.

Rapid tool for identification of bacterial strains using Fourier transform infrared spectroscopy on genomic DNA.

AIMS: We developed a new rapid and reliable method for identifying bacteria using a combination of Fourier transform infrared (FT-IR) spectroscopy of bacterial genomic DNA and multivariate analysis. METHODS AND RESULTS: FT-IR spectra of genomic DNA from four type strains of Pseudomonas spp., three type strains of Escherichia spp. and two type strains of Bacillus spp. were analysed in the 4000-400 cm-1 region. Spectral differences were found in the frequency regions of N-H stretching (amide I), C=O stretching vibrations (amide II) and PO2 - ionized asymmetric and symmetric stretching. Partial least squares discriminant analysis of the FT-IR spectra showed that the microbial strains could be discriminated by hierarchical clustering analysis. CONCLUSIONS: FT-IR spectral analysis of bacterial genomic DNA has potential for the rapid identification of bacteria at the genus and species levels. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports a new bacterial identification method using multivariate analysis of FT-IR spectra of bacterial genomic DNA.

The mating pilus of E. coli pED208 acts as a conduit for ssDNA during horizontal gene transfer.

IMPORTANCE: Bacteria are constantly exchanging DNA, which constitutes horizontal gene transfer. While some of these occurs by a non-specific process called natural transformation, some occurs by a specific mating between a donor and a recipient cell. In specific conjugation, the mating pilus is extended from the donor cell to make contact with the recipient cell, but whether DNA is actually transferred through this pilus or by another mechanism involving the type IV secretion system complex without the pilus has been an open question. Using Escherichia coli, we show that DNA can be transferred through this pilus between a donor and a recipient cell that has not established a tight mating junction, providing a new picture for the role of this pilus.

Assembly of respiratory syncytial virus matrix protein lattice and its coordination with fusion glycoprotein trimers.

Respiratory syncytial virus (RSV) is an enveloped, filamentous, negative-strand RNA virus that causes significant respiratory illness worldwide. RSV vaccines are available, however there is still significant need for research to support the development of vaccines and therapeutics against RSV and related Mononegavirales viruses. Individual virions vary in size, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 µm in length. Though the general arrangement of structural proteins in virions is known, we use cryo-electron tomography and sub-tomogram averaging to determine the molecular organization of RSV structural proteins. We show that the peripheral membrane-associated RSV matrix (M) protein is arranged in a packed helical-like lattice of M-dimers. We report that RSV F glycoprotein is frequently observed as pairs of trimers oriented in an anti-parallel conformation to support potential interactions between trimers. Our sub-tomogram averages indicate the positioning of F-trimer pairs is correlated with the underlying M lattice. These results provide insight into RSV virion organization and may aid in the development of RSV vaccines and anti-viral targets.

Broad protection against clade 1 sarbecoviruses after a single immunization with cocktail spike-protein-nanoparticle vaccine.

The 2002 SARS outbreak, the 2019 emergence of COVID-19, and the continuing evolution of immune-evading SARS-CoV-2 variants together highlight the need for a broadly protective vaccine against ACE2-utilizing sarbecoviruses. While updated variant-matched formulations are a step in the right direction, protection needs to extend beyond SARS-CoV-2 and its variants to include SARS-like viruses. Here, we introduce bivalent and trivalent vaccine formulations using our spike protein nanoparticle platform that completely protect female hamsters against BA.5 and XBB.1 challenges with no detectable virus in the lungs. The trivalent cocktails elicit highly neutralizing responses against all tested Omicron variants and the bat sarbecoviruses SHC014 and WIV1. Finally, our 614D/SHC014/XBB trivalent spike formulation completely protects human ACE2-transgenic female hamsters against challenges with WIV1 and SHC014 with no detectable virus in the lungs. Collectively, these results illustrate that our trivalent protein-nanoparticle cocktail can provide broad protection against SARS-CoV-2-like and SARS-CoV-1-like sarbecoviruses.

CCL11 released by GSDMD-mediated macrophage pyroptosis regulates angiogenesis after hindlimb ischemia.

Peripheral vascular disease (PVD) is an emerging public health burden with a high rate of disability and mortality. Gasdermin D (GSDMD) has been reported to exert pyroptosis and play a critical role in the pathophysiology of many cardiovascular diseases. We ought to determine the role of GSDMD in the regulation of perfusion recovery after hindlimb ischemia (HLI). Our study revealed that GSDMD-mediated pyroptosis occurred in HLI. GSDMD deletion aggravated perfusion recovery and angiogenesis in vitro and in vivo. However, how GSDMD regulates angiogenesis after ischemic injury remains unclear. We then found that GSDMD-mediated pyroptosis exerted the angiogenic capacity in macrophages rather than endothelial cells after HLI. GSDMD deletion led to a lower level of CCL11 in mice serum. GSDMD knockdown in macrophages downregulated the expression and decreased the releasing level of CCL11. Furthermore, recombinant CCL11 improved endothelial functions and angiogenesis, which was attenuated by CCL11 antibody. Taken together, these results demonstrate that GSDMD promotes angiogenesis by releasing CCL11, thereby improving blood flow perfusion recovery after hindlimb ischemic injury. Therefore, CCL11 may be a novel target for prevention and treatment of vascular ischemic diseases.

Comparison of the effects of dexmedetomidine, ketamine, and placebo on emergence agitation after strabismus surgery in children.

BACKGROUND: Children undergoing strabismus surgery under sevoflurane anesthesia often experience emergence agitation (EA) and postoperative vomiting (POV). This study compared the effects of intraoperative dexmedetomidine, ketamine, and placebo on postoperative EA and POV. METHODS: Eighty-four children (aged two to seven years) undergoing elective strabismus surgery under sevoflurane anesthesia were randomly assigned to one of three groups (n = 28 each). Intraoperatively, the placebo, dexmedetomidine, and ketamine groups received normal saline, dexmedetomidine 1 µg·kg(-1) iv plus a 1 µg·kg(-1)·hr(-1) infusion, and ketamine 1 mg·kg(-1) iv plus a 1 mg·kg(-1)·hr(-1) infusion, respectively. Agitation scores (Pediatric Anesthesia Emergence Delirium [PAED] scale) and POV were assessed in the postanesthetic care unit (PACU) and for 24 hr on the ward. Pain scores and times to laryngeal mask airway (LMA™) removal, resumption of mental orientation, and discharge from the PACU were also assessed. RESULTS: Seventy-eight children completed the study. Peak PAED scores for EA were lower in the dexmedetomidine (P < 0.001) and ketamine (P = 0.002) groups than in the placebo group. Incidence of POV was lower in the dexmedetomidine group (15%) than in the ketamine (44%; P = 0.02) or placebo (45.8%; P = 0.02) groups. Pain scores on the ward were lower in the dexmedetomidine (P < 0.001) and ketamine (P < 0.001) groups than in the placebo group. Time to LMA removal was similar in all groups. Time for resumption of mental orientation and time to discharge from PACU were longer in the dexmedetomidine and ketamine groups than in the placebo group. CONCLUSIONS: Dexmedetomidine and ketamine appear to prevent postoperative agitation and pain after sevoflurane anesthesia for pediatric strabismus surgery. Dexmedetomidine also prevents POV.

Atomic-level architecture of Caulobacter crescentus flagellar filaments provide evidence for multi-flagellin filament stabilization.

Flagella are dynamic, ion-powered machines with assembly pathways that are optimized for efficient flagella production. In bacteria, dozens of genes are coordinated at specific times in the cell lifecycle to generate each component of the flagellum. This is the case for Caulobacter crescentus, but little is known about why this species encodes six different flagellin genes. Furthermore, little is known about the benefits multi-flagellin species possess over single flagellin species, if any, or what molecular properties allow for multi-flagellin filaments to assemble. Here we present an in-depth analysis of several single flagellin filaments from C. crescentus, including an extremely well-resolved structure of a bacterial flagellar filament. We highlight key molecular interactions that differ between each bacterial strain and speculate how these interactions may alleviate or impose helical strain on the overall architecture of the filament. We detail conserved residues within the flagellin subunit that allow for the synthesis of multi-flagellin filaments. We further comment on how these molecular differences impact bacterial motility and highlight how no single flagellin filament achieves wild-type levels of motility, suggesting C. crescentus has evolved to produce a filament optimized for motility comprised of six flagellins. Finally, we highlight an ordered arrangement of glycosylation sites on the surface of the filaments and speculate how these sites may protect the ß-hairpin located on the surface exposed domain of the flagellin subunit.

Comparison of different weight-based scalars of remimazolam tosylate for anesthesia induction in obese patients: study protocol for a prospective, controlled trial.

BACKGROUND: The physiologic and anthropometric characteristics changes associated with obesity may result in the alternation of pharmacologic management. Remimazolam tosylate is a new type of ultra-short-acting benzodiazepine with stable context-sensitive half-time (CSHT) and no lipid accumulation after long-time infusion. Although remimazolam tosylate has potential advantages for the induction and maintenance of anesthesia in obese patients, the appropriate induction dosing scalars among obese patients are unknown. Therefore, we aim to compare the different weight-based scalars for dosing remimazolam tosylate of anesthesia induction among obese patients. METHODS/DESIGN: The study will be performed as a prospective, single-center, double-blind, controlled clinical trial. The study design is a comparison of remimazolam tosylate requirements based on total body weight (TBW) or lean body weight (LBW) to reach a Modified Observer's Assessment of Alertness and Sedation (MOAA/S) score of 0 among obese subjects (BMI ≥ 35 kg/m2). Another twenty normal-weight subjects (18.5 kg/m2 ≤ BMI < 25 kg/m2) will be enrolled as a control group, whose induction dose is scaled based on TBW. The infusion rate of remimazolam tosylate during induction is 12 mg/kg/h in all groups. DISCUSSION: Results of the present study will provide evidence of dose scalar of remimazolam tosylate to guide the clinical practice of anesthesia induction in obese patients. TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR220005664. Registered on 9 February 2022, https://www.chictr.org.cn/showproj.aspx?proj=151150 .

Handling difficult cryo-ET samples: A study with primary neurons from Drosophila melanogaster.

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

Galangin alleviated myocardial ischemia-reperfusion injury by enhancing autophagic flux and inhibiting inflammation.

Autophagy is critically involved in myocardial ischemia-reperfusion (I/R). Autophagy inhibition exacerbates myocardial I/R injury. Few effective agents target autophagy to prevent myocardial I/R injury. Effective drugs that promote autophagy in myocardial I/R warrant further investigation. Galangin (Gal) enhances autophagy and alleviates I/R injury. Here we conducted both in vivo and in vitro experiments to observe the changes in autophagy after galangin treatment and investigated the cardioprotective effects of galangin on myocardial I/R. METHODS: After 45-min occlusion of the left anterior descending coronary artery, myocardial I/R was induced by slipknot release. One day before surgery and immediately after surgery, the mice were injected intraperitoneally with the same volume of saline or Gal. The effects of Gal were evaluated using echocardiography, 2,3,5-triphenyltetrazolium chloride staining (TTC staining), western blotting, and transmission electron microscopy. Primary cardiomyocytes and bone marrow-derived macrophages were extracted in vitro to measure the cardioprotective effects of Gal. RESULTS: Compared with the saline-treated group, Gal significantly improved cardiac function and limited infarct enlargement after myocardial I/R. In vivo and in vitro studies demonstrated that Gal treatment promoted autophagy during myocardial I/R. The anti-inflammatory effects of Gal were validated in bone marrow-derived macrophages. These results strongly suggest that Gal treatment can attenuate myocardial I/R injury. CONCLUSION: Our data demonstrated that Gal could improve left ventricular ejection fraction and reduce infarct size after myocardial I/R by promoting autophagy and inhibiting inflammation.

Perioperative Low Dose Dexmedetomidine and Its Effect on the Visibility of the Surgical Field for Middle Ear Microsurgery: A Randomised Controlled Trial.

Background and Purpose: There are many benefits of administering dexmedetomidine perioperatively. The pharmacokinetics (PK) and pharmacodynamics (PD) of intravenous, intranasal and oral dexmedetomidine that was administered before anesthesia were compared in this study, and the effects of dexmedetomidine on the surgical field visibility in tympanoplasty was evaluated. Methods: A single-blind, randomized controlled trial was conducted in a university-affiliated hospital where 45 patients who underwent tympanoplasty under general anesthesia were randomly allocated into three groups. Dexmedetomidine was administered by intravenous infusion at 0.8 µg.kg-1 for 10 min, intranasal instillation at a drop rate of 1 µg.kg-1 and oral intake at 4 µg.kg-1 ten minutes before the induction of anesthesia. The PK and PD of dexmedetomidine after a single low dose administration and its effect on the surgical field in tympanoplasty were analysed. Results: A plasma concentration of dexmedetomidine of 220 pg/ml was achieved immediately after intravenous infusion and at 13.2 and 70.3 min for intranasal and oral administration, respectively. Dexmedetomidine decreased the heart rate (HR) and mean arterial pressure (MAP) in all three groups, although these values remained higher in the oral dexmedetomidine group at all eight time points. Intravenous dexmedetomidine provided the best visualization of the surgical field for opening of the tympanic sinus, 30 min after the start of the infusion (p < 0.05). Intranasal dexmedetomidine provided a significantly better visual field than oral dexmedetomidine for the repair of a tympanic membrane perforation using the fascia temporal muscle (p < 0.05). Conclusion: A single low dose of dexmedetomidine administered intravenously or intranasally could decrease HR and MAP, improve surgical field visibility and be appropriate for deliberate hypotension for surgical procedures of 1-2 h in length. Trial registration: Clinicaltrials.gov identifier: NCT03800641.

Research Progress on Risk Factors of Preoperative Anxiety in Children: A Scoping Review.

BACKGROUND: Preoperative anxiety has adverse effects on children and negative impacts on postoperative rehabilitation. Anesthesiologists can accurately identify children with preoperative anxiety, and individualized intervention can effectively improve their psychological state and clinical prognosis. However, a comprehensive summary of the current available evidence has yet to be conducted. Searches were conducted in Medline databases from inception to March 2022. Primary studies that reported preoperative anxiety in children and its attendant effects on postoperative recovery and prognosis were screened and included. Among the 309 publications identified, 12 related studies (n = 3540 patients) met the eligibility criteria. The incidence of preoperative anxiety in children in the included studies ranged from 41.7% to 75.44%. While 16 influencing factors were identified, only 5 factors had a significant impact on preoperative anxiety in children: younger age (n = 8), parental anxiety (n = 7), negative previous hospitalizations (n = 3), less sociableness (n = 2), and surgical setting (n = 1). The current scoping review identified risk factors for preoperative anxiety in children. Healthcare workers should identify and manage preoperatively anxious children. There are still some factors that are controversial, and large-scale clinical studies are needed.