Role of the tonsil-oropharynx ratio on lateral cephalograms in assessing tonsillar hypertrophy in children seeking orthodontic treatment.

OBJECTIVES: To analyze the diagnostic value of the tonsil-oropharynx (T/O) ratio on lateral cephalograms for evaluating tonsillar hypertrophy (TH). METHODS: A cross-sectional study was performed on 185 consecutive children (101 males, 84 females; mean age 7.3 ± 1.4 years) seeking orthodontic treatment. The T/O ratios on lateral cephalograms were calculated following Baroni et al.'s method. Tonsil sizes were clinically determined according to the Brodsky grading scale. Spearman correlation coefficients between the T/O ratio and clinical tonsil size were calculated with the total sample and subgroups and then compared between subgroups. Diagnostic value was analyzed using the receiver operating characteristic (ROC) curve, sensitivity, specificity, positive and negative predictive values, and accuracy. RESULTS: There was a strong correlation between the T/O ratio and clinical tonsil size in children (ρ = 0.73; P < 0.001). A significantly higher correlation coefficient was found in the Class III children. The ROC curve revealed an area under the curve of 0.90 (95% CI, 0.86-0.94; P < 0.001). The optimal cutoff value of the T/O ratio for predicting TH was 0.58, with a sensitivity of 98.7% and specificity of 64.2%. Employing the cutoff value of 0.5, the sensitivity was 100% and the specificity was 45.9%. CONCLUSIONS: Measurement of the T/O ratio on lateral cephalograms may be helpful to initial screening in children for TH. Practitioners may combine the clinical examination of tonsil size with the cephalometric findings for a more comprehensive evaluation.

Effects of Maxillary Skeletal Expansion on Upper Airway Airflow: A Computational Fluid Dynamics Analysis.

The effect of maxillary skeletal expansion (MSE) on upper airway in adolescent patients is not clear. The purpose of this study was to determine the upper airway airflow with MSE treatment using computational fluid dynamics analysis. Three-dimensional upper airway finite element models fabricated from cone beam computed tomography images were obtained before and after treatment in an adolescent patient with maxillary constriction. Turbulent analyses were applied. The nasal cavity (NC) was divided into 6 planes along the y-axis and the pharynx was divided into 7 planes in the z-axis. Changes in cross-sectional area, airflow velocity, pressure, and total resistance at maximum expiration and maximum inspiration were determined at each plane after MSE treatment. The greatest increase in area occurred in the oropharynx which was around 40.65%. The average increase in area was 7.42% in the NC and 22.04% in the pharynx. The middle part of pharynx showed the greatest increase of 212.81 mm and 217.99 mm or 36.58% and 40.66%, respectively. During both inspiration and expiration, airflow pressure decreased in both the NC and pharynx, which ranged from -11.34% to -23.68%. In the NC, the average velocity decrease was -0.18 m/s at maximum expiration (ME) and -0.13 m/s at maximum inspiration (MI). In the pharynx, the average velocity decrease was -0.07 m/s for both ME and MI. These results suggest that treatment of maxillary constriction using MSE appliance may show positive effects in improvement of upper airway cross-sectional areas and reduction of upper airway resistance and velocity.

RASSF4 promotes EV71 replication to accelerate the inhibition of the phosphorylation of AKT.

Enterovirus 71 (EV71) is a neurotropic virus that causes hand, foot and mouth disease (HFMD), occasionally leading to death. As a member of the RAS association domain family (RASSFs), RASSF4 plays important roles in cell death, tumor development and signal transduction. However, little is known about the relationship between RASSF4 and EV71. Our study reveals for the first time that RASSF4 promotes EV71 replication and then accelerates AKT phosphorylation inhibition in EV71-infected 293T cells, suggesting that RASSF4 may be a potential new target for designing therapeutic measures to prevent and control EV71 infection.

miR-27a suppresses EV71 replication by directly targeting EGFR.

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, has broken out several times and was accompanied by neurological disease. microRNAs, a class of small non-coding RNAs that are approximately 20 nucleotides long, play important roles in the regulation of various biological processes, including antiviral defense. However, the roles of miRNAs in EV71 replication and pathogenesis are not well understood. In this study, we found that the expression of miR-27a was significantly decreased in EV71-infected cells. Interestingly, the over-expression of miR-27a could inhibit EV71 replication, as measured by virus titration, qPCR, and Western blotting. We identified EGFR mRNA is a bona fide target of miR-27a by computational analysis and luciferase reporter assays. Furthermore, miR-27a could decrease EGFR expression, as measured by qPCR and Western blotting. Moreover, the inhibition of EGFR expression by miR-27a decreased the phosphorylation of Akt and ERK, which facilitate EV71 replication. These results suggest that miR-27a may have antiviral activity against EV71 by inhibiting EGFR.

An array of femtoliter wells for sensitive detection of copper using click chemistry.

Sensitive detection of copper ion (Cu2+), which is of great importance for environmental pollution and human health, is crucial. In this study, we present a highly sensitive method for measuring Cu2+ in an array of femtoliter wells. In brief, magnetic beads (MBs) modified with alkyne groups were bound to the azide groups of biotin-PEG3-azide (bio-PEG-N3) via Cu+-catalyzed click chemistry. Cu+ in the click chemistry reaction was generated by reducing Cu2+ with sodium ascorbate. Following the ligation, the surface of the MBs was modified with biotin, which could be labeled with streptavidin-ß-galactosidase (SßG). The MBs complex was then suspended in ß-galactosidase substrate fluorescein-di-ß-d-galactopyranoside (FDG), and loaded into the array of femtoliter wells. The MBs sank into the wells due to gravity, and the resulting fluorescent product, generated from the reaction between SßG on the surface of the MBs and FDG, was confined within the wells. The number of fluorescent wells increased with higher Cu2+ concentrations. The bright-field and fluorescent images of the wells were acquired using an inverted fluorescent microscope. The detection limit of this assay for Cu2+ was 1 nM without signal amplification, which was 103 times lower than that of traditional fluorescence detection assays.

Impact of tonsillectomy on the efficacy of Alt-RAMEC/PFM treatment protocols in children with class III malocclusion and tonsillar hypertrophy: protocol for a cluster randomised controlled trial.

INTRODUCTION: Orthodontic treatment using face mask protraction combined with an alternate rapid maxillary expansion and constriction/protraction face mask (Alt-RAMEC/PFM) protocol is effective in the early treatment of patients with class III malocclusion, but the stability of treatment outcomes represents a major concern. Previous studies have suggested that tonsillar hypertrophy can be a risk factor for class III malocclusion and tonsillectomy may prompt the normalisation of dentofacial growth. However, these studies had a low-to-moderate level of evidence. This study was designed to identify the impact of tonsillectomy before orthodontic treatment on the efficacy and stability of Alt-RAMEC/PFM protocols and the sleep quality and oral health in children with anterior crossbite and tonsillar hypertrophy. METHODS AND ANALYSIS: This is a two-arm, parallel-group, superiority cluster randomised controlled trial, with four clinics randomly assigned to the surgery-first arm and the orthodontic-first arm in a 1:1 ratio. The Alt-RAMEC protocol involves alternate activation and deactivation of the expander's jet screw over 6 weeks to stimulate maxillary suture distraction. Patients will be instructed to wear the PFM for a minimum of 14 hours per day. The primary outcomes are changes in Wits appraisal and the degree of maxillary advancement from baseline to the end of orthodontic treatment. Lateral cephalometric radiographs, polysomnography, Obstructive Sleep Apnoea-18 questionnaire and Oral Health Impact Profile-14 questionnaire will be traced, collected and measured. We will recruit 96 patients intofor the study. To assess differences, repeated multilevel linear mixed modelling analyses will be used. ETHICS AND DISSEMINATION: This study has been granted ethical approval by the Ethics Committee of the School & Hospital of Stomatology, Wuhan University (approval No. 2023-D10). Written informed consent will be obtained from the participants and their guardians. The results of the trial will be disseminated through academic conferences and journal publications. TRIAL REGISTRATION NUMBER: ChiCTR2300078833.

Xylose recovery and bioethanol production from sugarcane bagasse pretreated by mild two-stage ultrasonic assisted dilute acid.

Pretreatment can improve biomass biodegradability. Here, a novel sugarcane bagasse (SCB) pretreatment process based on two-stage ultrasonic assisted dilute H2SO4 (TUDA) under mild conditions was reported. After optimization, the pretreatment was shown to significantly degrade hemicellulose (92.40%) and remove lignin (57.41%) of SCB, leading to reduction of inhibitors and an ethanol fermentation efficiency of 93.37% by SSCF under cellulase 10 FPU/g SCB and 30% pretreated SCB loading. Physical characterization revealed that two-stage ultrasonic could better disrupt SCB than traditional ultrasonic by amplifying the collapse effect and synergistically promoting lignin removal through dilute H2SO4. Furthermore, xylose was also effectively recovered from pretreatment supernatant by biochar derived from bagasse. This study established a simple and efficient pretreatment process for high value-added recycling of SCB from solid residue to pretreatment liquid.

Bioaugmentation combined with biochar to enhance thermophilic hydrogen production from sugarcane bagasse.

In this study, Thermoanaerobacterium thermosaccharolyticum MJ2 and biochar were used to enhance thermophilic hydrogen production from sugarcane bagasse. MJ2 bioaugmentation notably increased the hydrogen production by 95.31%, which was further significantly improved by 158.10% by adding biochar. The addition of biochar promoted the degradation of substrate, improved the activities of hydrogenase and electron transfer system, and stimulated microbial growth and metabolism. Microbial community analysis showed that the relative abundance of Thermoanaerobacterium was significantly increased by bioaugmentation and further enriched by biochar. PICRUSt analysis showed that MJ2 combined with biochar promoted metabolic pathways related to substrate degradation and microbial metabolism. This study provides a novel enhancement method for hydrogen production of the cellulolytic microbial consortium by exogenous hydrogen-producing microorganism combined with biochar and deepens the understanding of its functional mechanism.

A combination of deep eutectic solvent and ethanol pretreatment for synergistic delignification and enhanced enzymatic hydrolysis for biorefinary process.

A novel pretreatment system containing deep eutectic solvents and ethanol (DES-E) for synergistic carbohydrate conversion and delignification was reported in this study. The DES-E pretreatment resulted in an enhanced glucose yield compared to individual DES and ethanol pretreatment for the three tested biomass, including Broussonetia papyrifera, corn stover and pine. To further explore the delignification mechanism, the solubilized lignin and residual lignin from Broussonetia papyrifera was recovered and extracted, then thoroughly characterized. The highest total OH content was found in the DES-E solubilized lignin, which could be used as antioxidant. The presence of ethanol in pretreatment liquor could protect the ß-O-4 substructure from breakage and reduce lignin condensation, which favors the subsequent enzymatic hydrolysis. Comparable glucose yield and delignification performance was achieved by recycled DES. DES-E pretreatment offers a promising method for lignin isolation and cellulose digestibility improvement simultaneously.

A conserved membrane attachment site in alpha-SNAP facilitates N-ethylmaleimide-sensitive factor (NSF)-driven SNARE complex disassembly.

The ATPase NSF (N-ethylmaleimide-sensitive factor) and its SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) cofactor constitute the ubiquitous enzymatic machinery responsible for recycling of the SNARE (SNAP receptor) membrane fusion machinery. The enzyme uses the energy of ATP hydrolysis to dissociate the constituents of the SNARE complex, which is formed during the fusion of a transport vesicle with the acceptor membrane. However, it is still unclear how NSF and the SNAP adaptor work together to take the tight SNARE bundle apart. SNAPs have been reported to attach to membranes independently from SNARE complex binding. We have investigated how efficient the disassembly of soluble and membrane-bound substrates are, comparing the two. We found that SNAPs support disassembly of membrane-bound SNARE complexes much more efficiently. Moreover, we identified a putative, conserved membrane attachment site in an extended loop within the N-terminal domain of alpha-SNAP. Mutation of two highly conserved, exposed phenylalanine residues on the extended loop prevent SNAPs from facilitating disassembly of membrane-bound SNARE complexes. This implies that the disassembly machinery is adapted to attack membrane-bound SNARE complexes, probably in their relaxed cis-configuration.

Preparation and characterization of a novel colorimetric indicator film based on gelatin/polyvinyl alcohol incorporating mulberry anthocyanin extracts for monitoring fish freshness.

This work aimed to develop a novel colorimetric indicator film for monitoring of food freshness based on gelatin/polyvinyl alcohol matrix incorporated with anthocyanin extracts from mulberry. The color of anthocyanin extracts solutions obviously changed from bright red to dark green in the pH range of 2.0-11.0. FTIR spectra and isothermal titration calorimetry showed that the anthocyanin extracts were successfully combined with gelatin/polyvinyl alcohol matrix by hydrogen binding and electrostatic interaction, which enhanced the stability of anthocyanin. The scanning electric microscopy showed that the compatibility between polyvinyl alcohol and gelatin were improved owing to the addition of anthocyanin extracts. With the anthocyanin extracts addition from 0 to 45 mg/100 mL mixed solution, the tensile strength decreased from 30.80 to 21.01 MPa and the elongation at break increased from 589.22% to 905.86%. The color response of film in buffer solution of different pH were in accordance with anthocyanin extracts solutions, and its color changes were clearly visible with naked eye. Finally, the film was evaluated by a test on monitoring fish spoilage, which presented visible color changes due to volatile nitrogenous compounds formed over time. These results showed that this developed film could be used as an effective method for the monitoring of food freshness.

[Clinical characteristics and diagnosis of rheumatoid arthritis of upper cervical spine: analysis of 71 cases].

OBJECTIVE: To study the clinical characteristics and diagnosis of rheumatoid arthritis (RA) in the upper cervical spine. METHODS: The clinical data of 71 patients with RA in the upper cervical spine, 18 males and 53 females, aged 46.2 (23-76), with a mean duration of RA of 18.2 years (2 months-47 years) were retrospectively reviewed. Fifty-three patients received glucocorticoid for more than 3 months. In addition to routine examinations, all patients underwent plain X-ray film taking, CT and MRI scanning, and erythrocyte sedimentation rate, rheumatoid factor (RF) and antistreptolysin O testing. RESULTS: The symptoms of upper cervical spine appeared at 8.3 years (2 months-46 years) after the diagnosis of RA was confirmed. The clinical manifestations of RA in the upper cervical spine were intractable pain in craniocervical junction or radiating pain. Abnormal postures in the neck and Sherp-Purser's sign were positive in some patients. Progressive neurological dysfunction with the involvement of spinal cord, medulla, or some cranial nerves might gradually appear. Irregular destruction of bone with osteoporosis around the lateral and median atlantoaxial joint was a common finding in the X-ray films and CT scans. Instability of the atlantoaxial joint, including anterior atlantoaxial subluxation, posterior atlantoaxial subluxation and anterior-posterior atlantoaxial subluxation were found in 68 cases, while rotation subluxation was presented in 37 cases. Vertical migration of the odontoid was seen in 11 cases. RF was positive in 18 cases. MRI revealed that the cause of spinal cord compression was the bone tissue and soft tissue pannus. CONCLUSIONS: RA in the upper cervical spine is a common situation in the clinical settings. The key point in the diagnosis of this disease is the identification of instability in the atlantoaxial joint and assessment of the spinal neurological deficit. And a careful analysis of the natural history will further help to achieve a better treatment effect.

BMAL1 Deficiency Contributes to Mandibular Dysplasia by Upregulating MMP3.

Skeletal mandibular hypoplasia (SMH), one of the common types of craniofacial deformities, seriously affects appearance, chewing, pronunciation, and breathing. Moreover, SMH is prone to inducing obstructive sleep apnea syndrome. We found that brain and muscle ARNT-like 1 (BMAL1), the core component of the molecular circadian oscillator, was significantly decreased in mandibles of juvenile SMH patients. Accordingly, SMH was observed in circadian-rhythm-disrupted or BMAL1-deficient mice. RNA sequencing and protein chip analyses suggested that matrix metallopeptidase 3 (MMP3) is the potential target of BMAL1. Interestingly, in juvenile SMH patients, we observed that MMP3 was obviously increased. Consistently, MMP3 was upregulated during the whole growth period of 3-10 weeks in Bmal1-/- mice. Given these findings, we set out to characterize the underlying mechanism and found BMAL1 deficiency enhanced Mmp3 transcription through activating p65 phosphorylation. Together, our results provide insight into the mechanism by which BMAL1 is implicated in the pathogenesis of SMH.

A new online exhaust gas monitoring system in hydrochloric acid regeneration of cold rolling mills.

Measuring the content of hydrogen chloride (HCl) in exhaust gas used to take time and energy. In this paper, we introduce a new online monitoring system which can output real-time data to the monitoring center. The system samples and cools exhaust gas, and after a series of processing, it will be analyzed by a specific instrument. The core part of this system is remote terminal unit (RTU) which is designed on Cortex-A8 embedded architecture. RTU runs a scaled-down version of Linux which is a good choice of OS for embedded applications. It controls the whole processes, does data acquisition and data analysis, and communicates with monitoring center through Ethernet. In addition, through a software developed for windows, the monitoring process can be remotely controlled. The new system is quite beneficial for steel industry to do environment monitoring.

Rapid prototyping technology and its application in bone tissue engineering.

Bone defects arising from a variety of reasons cannot be treated effectively without bone tissue reconstruction. Autografts and allografts have been used in clinical application for some time, but they have disadvantages. With the inherent drawback in the precision and reproducibility of conventional scaffold fabrication techniques, the results of bone surgery may not be ideal. This is despite the introduction of bone tissue engineering which provides a powerful approach for bone repair. Rapid prototyping technologies have emerged as an alternative and have been widely used in bone tissue engineering, enhancing bone tissue regeneration in terms of mechanical strength, pore geometry, and bioactive factors, and overcoming some of the disadvantages of conventional technologies. This review focuses on the basic principles and characteristics of various fabrication technologies, such as stereolithography, selective laser sintering, and fused deposition modeling, and reviews the application of rapid prototyping techniques to scaffolds for bone tissue engineering. In the near future, the use of scaffolds for bone tissue engineering prepared by rapid prototyping technology might be an effective therapeutic strategy for bone defects.

Identification and characterization of the abscisic acid (ABA) receptor gene family and its expression in response to hormones in the rubber tree.

Abscisic acid (ABA) is an essential phytohormone involved in diverse physiological processes. Although genome-wide analyses of the ABA receptor PYR/PYL/RCAR (PYL) protein/gene family have been performed in certain plant species, little is known about the ABA receptor protein/gene family in the rubber tree (Hevea brasiliensis). In this study, we identified 14 ABA receptor PYL proteins/genes (designated HbPYL1 through HbPYL14) in the most recent rubber tree genome. A phylogenetic tree was constructed, which demonstrated that HbPYLs can be divided into three subfamilies that correlate well with the corresponding Arabidopsis subfamilies. Eight HbPYLs are highly expressed in laticifers. Five of the eight genes are simultaneously regulated by ABA, jasmonic acid (JA) and ethylene (ET). The identification and characterization of HbPYLs should enable us to further understand the role of ABA signal in the rubber tree.

The long non-coding RNA expression profile of Coxsackievirus A16 infected RD cells identified by RNA-seq.

Coxsackievirus A16 (CVA16) is one of major pathogens of hand, foot and mouth disease (HFMD) in children. Long non-coding RNAs (IncRNAs) have been implicated in various biological processes, but they have not been associated with CVA16 infection. In this study, we comprehensively characterized the landscape of IncRNAs of normal and CVA16 infected rhabdomyosarcoma (RD) cells using RNA-Seq to investigate the functional relevance of IncRNAs. We showed that a total of 760 IncRNAs were upregulated and 1210 IncRNAs were downregulated. Out of these dysregulated IncRNAs, 43.64% were intergenic, 22.31% were sense, 15.89% were intronic, 8.67% were bidirectional, 5.59% were antisense, 3.85% were sRNA host IncRNAs and 0.05% were enhancer. Six dysregulated IncRNAs were validated by quantitative PCR assays and the secondary structures of these IncRNAs were projected. Moreover, we conducted a bioinformatics analysis of an IncRNAs (ENST00000602478) to elucidate the diversity of modification and functions of IncRNAs. In summary, the current study compared the dysregulated IncRNAs profile upon CVA16 challenge and illustrated the intricate relationship between coding and IncRNAs transcripts. These results may not only provide a complete picture of transcription in CVA16 infected cells but also provide novel molecular targets for treatments of HFMD.

[Improving the surgical technology in treatment of OSAHS].

Noninvasive continuous positive airway pressure (CPAP) for the ventilation treatment of obstructive sleep apnea-hypopnea syndrome (OSAHS) is the first-line treatment method. This article aims to describe the effect of surgical operation especially for the UPPP in the individualized comprehensive treatment of OSAHS and the importance of surgical technique. Lower compliance is the bottleneck of CPAP therapy in clinical application, for the OSAHS patients with treatment failure in CPAP or those cannot accept CPAP therapy, when with no other ideal instrument therapeutics, accurate diagnosis of position in airway obstruction with an adequate surgical operation is the treatment,of choice. Surgical operation is particularly important either as a fore-lying means to improve the CPAP treatment compliance or as an independence treatment method of OSAHS. The pharyngeal cavity is the most common obstructive plane in patients with OSAHS. The operation of traditional UPPP aiming at the expansion of pharyngeal cavity is the classics surgery to solve obstruction in this plane, the lower operation effective rate is the main reason of restriction in its development. How to improve the effective rate of surgical treatment of OSAHS is our surgical goal. The effective rate of surgical operation treatment in OSAHS rely on the following sides: to follow the OSAHS individualized comprehensive treatment principle, reasonable choice of surgical operation indication, the precise localization diagnosis of upper airway obstruction, adequate surgical operation and skilled surgical techniques.

Effects of polymorphs on dissolution of cellulose in NaOH/urea aqueous solution.

This study focused on the effect of cellulose crystal type on the dissolution of cellulose in aqueous NaOH/urea. Cellulose I, II, IIII, and IVI were prepared and characterized. Subsequently, the solubility of these cellulose samples in aqueous NaOH/urea was tested and the mechanism was determined by a combination of (1)H NMR spectroscopy and differential scanning calorimetry (DSC). The results indicated that, compared with the degree of polymerization (DP) and crystallinity index of cellulose, the cellulose crystal type had greater impact on cellulose dissolution. Specially, the solubility of the cellulose I, II, and III was progressively decreased, probably due to different hydrogen bond network and packing energy in it. The ability of cellulose I, II, III, and IV to form hydrogen bonds with sodium hydroxides was nearly the same, while gradually increased when cellulose preparations were associated with urea. Moreover, all the cellulose solutions showed good thermal stability.

[Comparation between modified coblation assisted uvulopalatopharyngoplasty and conventional uvulopalatopharyngoplasty in operative complication].

OBJECTIVE: To compare the operative effectivity and incidence of postoperative complication between conventional uvulopalatopharyngoplasty (UPPP) and modified coblation assisted UPPP (M-CAUP) in treating patients with obstructive sleep apnea-hypopnea syndrome (OSAHS). And to explore the more effective, safer and minimally invasive operative method in surgical therapy of OSAHS. METHODS: It was a controlled trial. A retrospective analysis was made on surgical complications of conventional UPPP and of M-CAUP performed on OSAHS patients from 1995 to 2010. There were 451 patients in UPPP group and 323 patients in M-CAUP group. χ(2) test and Fisher's Exact test were used in statistical analysis. RESULTS: The incidence of serious surgical complications was higher in conventional UPPP group (3.77%, 17/451) than that in M-CAUP group (0.62%, 2/323), χ(2) = 7.800, P < 0.01, while the incidence of short-term complications was higher in M-CAUP group (90.40%, 292/323) than that in conventional UPPP group (60.98%, 275/451), χ(2) = 83.186, P < 0.01. The difference of long-term complications was not statistically significant between M-CAUP group and conventional UPPP group (P = 0.1331, Fisher Exact test). There was no significant difference in incidence of asphyxia between M-CAUP group and conventional UPPP group (P < 0.01, Fisher Exact test). However, the incidence of post-operative primary hemorrhage was obviously lower in M-CAUP group than that in conventional UPPP group (3.99%, 18/451), χ(2) = 12.133, P < 0.01. While the incidence of delayed hemorrhage, temporal velopharyngeal insufficiency, and foreign body sensation at pharynx were higher in M-CAUP group (8.05%, 12.69%, 68.42%, respectively) than that in conventional UPPP group (3.77%, 3.33%, 51.00%, respectively) P < 0.01, respectively. There was no significant difference in incidence of permanent velopharyngeal insufficiency, stenosis of nasopharynx and nasopharyngeal atresia, alteration of taste, throat itch and coughing. CONCLUSION: Compared to the conventional UPPP, M-CAUP was more effective and safer in treating OSAHS with less severe complications during and after the operation.