Bacteriophage and endolysin engineering for biocontrol of food pathogens/pathogens in the food: recent advances and future trends.

Despite advances in modern technologies, various foodborne outbreaks have continuously threatened the food safety. The overuse of and abuse/misuse of antibiotics have escalated this threat due to the prevalence of multidrug-resistant (MDR) pathogens. Therefore, the development of new methodologies for controlling microbial contamination is extremely important to ensure the food safety. As an alternative to antibiotics, bacteriophages(phages) and derived endolysins have been proposed as novel, effective, and safe antimicrobial agents and applied for the prevention and/or eradication of bacterial contaminants even in foods and food processing facilities. In this review, we describe recent genetic and protein engineering tools for phages and endolysins. The major aim of engineering is to overcome limitations such as a narrow host range, low antimicrobial activity, and low stability of phages and endolysins. Phage engineering also aims to deter the emergence of phage resistance. In the case of endolysin engineering, enhanced antibacterial ability against Gram-negative and Gram-positive bacteria is another important goal. Here, we summarize the successful studies of phages and endolysins treatment in different types of food. Moreover, this review highlights the recent advances in engineering techniques for phages and endolysins, discusses existing challenges, and suggests technical opportunities for further development, especially in terms of antimicrobial agents in the food industry.

In vivo measurement of trabecular meshwork stiffness in a corticosteroid-induced ocular hypertensive mouse model.

Ocular corticosteroids are commonly used clinically. Unfortunately, their administration frequently leads to ocular hypertension, i.e., elevated intraocular pressure (IOP), which, in turn, can progress to a form of glaucoma known as steroid-induced glaucoma. The pathophysiology of this condition is poorly understood yet shares similarities with the most common form of glaucoma. Using nanotechnology, we created a mouse model of corticosteroid-induced ocular hypertension. This model functionally and morphologically resembles human ocular hypertension, having titratable, robust, and sustained IOPs caused by increased resistance to aqueous humor outflow. Using this model, we then interrogated the biomechanical properties of the trabecular meshwork (TM), including the inner wall of Schlemm's canal (SC), tissues known to strongly influence IOP and to be altered in other forms of glaucoma. Specifically, using spectral domain optical coherence tomography, we observed that SC in corticosteroid-treated mice was more resistant to collapse at elevated IOPs, reflecting increased TM stiffness determined by inverse finite element modeling. Our noninvasive approach to monitoring TM stiffness in vivo is applicable to other forms of glaucoma and has significant potential to monitor TM function and thus positively affect the clinical care of glaucoma, the leading cause of irreversible blindness worldwide.

In vivo estimation of murine iris stiffness using finite element modeling.

The iris plays an important role in certain types of glaucoma, including primary angle-closure glaucoma and pigmentary glaucoma. Iris mechanics are also important in influencing trabecular meshwork deformation in response to intraocular pressure changes in some animal species. Although mice are widely used to study ocular disease, including glaucoma, the in vivo biomechanical properties of the murine iris are unknown. Thus, the primary objective of this study was to estimate murine iris biomechanical stiffness. We used optical coherence tomography (OCT) images of the anterior segment of living mice (n = 13, age = 7.3 ± 3.2 [mean ± SD] months) at sequentially increasing IOP levels, observing IOP-dependent iris deformations. We then used an inverse finite element model to predict iris deformations under the same conditions, estimating iris stiffness by maximizing agreement between OCT data and numerical simulations. Our results show an in vivo murine iris stiffness of 96.1 ± 54.7 kPa (mean ± SD), which did not correlate with age but was dependent on gender. Our results further showed strong evidence of reverse pupillary block, with mean posterior chamber pressure remaining at approximately 12 mmHg even as anterior chamber pressure was set to much higher levels. Our approach to monitoring iris stiffness in vivo is applicable to study potential changes of iris stiffness in various pathophysiological conditions and thus has significant potential for clinical care of ocular disease involving iris biomechanics.

Integral role for lysyl oxidase-like-1 in conventional outflow tissue function and behavior.

Lysyl oxidase-like-1 (LOXL1), a vital crosslinking enzyme in elastin fiber maintenance, is essential for the stability and strength of elastic vessels and tissues. Variants in the LOXL1 locus associate with a dramatic increase in risk of exfoliation syndrome (XFS), a systemic fibrillopathy, which often presents with ocular hypertension and exfoliation glaucoma (XFG). We examined the role of LOXL1 in conventional outflow function, the prime regulator of intraocular pressure (IOP). Using Loxl1-/- , Loxl1+/- , and Loxl1+/+ mice, we observed an inverse relationship between LOXL1 expression and IOP, which worsened with age. Elevated IOP in Loxl1-/- mice was associated with a larger globe, decreased ocular compliance, increased outflow facility, extracellular matrix (ECM) abnormalities, and dilated intrascleral veins, yet, no dilation of arteries or capillaries. Interestingly, in living Loxl1-/- mouse eyes, Schlemm's canal (SC) was less susceptible to collapse when challenged with acute elevations in IOP, suggesting elevated episcleral venous pressure (EVP). Thus, LOXL1 expression is required for normal IOP control, while ablation results in altered ECM repair/homeostasis and conventional outflow physiology. Dilation of SC and distal veins, but not arteries, is consistent with key structural and functional roles for elastin in low-pressure vessels subjected to cyclical mechanical stress.

Alcohol/Illicit Substance Use in Fatal Motorcycle Crashes.

BACKGROUND: The objective of the current study is to determine how alcohol and illicit substance use contributes to motorcycle crash fatalities by examining the relationship between toxicology levels found postmortem and the behavior of riders and passengers in fatal motorcycle crashes. MATERIALS AND METHODS: All motorcycle fatalities in Miami-Dade County, FL, from 2009 to 2014 were reviewed using the Miami-Dade County Medical Examiner's toxicology reports and the corresponding crash reports. RESULTS: Positive alcohol/illicit substance detection was found in 44% of our population of 227 fatalities. When compared with those with a negative alcohol/illicit substance detection, those with a positive alcohol/illicit substance detection were more likely to be found at fault of the crash (77% versus 50%, P < 0.001), more likely to be in a single-vehicle crash (47% versus 21%, P < 0.001) and less likely to wear a helmet (44% versus 64%, P = 0.002). However, there was no significant relationship between speeding and alcohol/illicit substance detection (29% versus 33%, P = 0.748). In addition, a regression analysis demonstrated that there was less helmet use and more single-vehicle crashes with higher blood alcohol concentration. CONCLUSIONS: In fatal motorcycle crashes, alcohol and illicit substance use had a significantly negative impact on the risk aversion of motorcycle fatalities in regard to fault, helmet use, and single-vehicle crashes.

Augmentation genioplasty using discarded bone fragments following proximal segment osteotomy of the ramus in intraoral vertical ramus osteotomy (IVRO).

BACKGROUND: Based on a three-dimensional (3D) orthognathic simulation, this technical report introduces a method for augmentation genioplasty using a proximal bone fragment of the mandible, which is typically discarded in intraoral vertical ramus osteotomy (IVRO). RESULTS: A 43-year-old female patient diagnosed with Class III malocclusion, presenting with a protruding mandible and long facial height, underwent surgical treatment. The surgical plan involved mandibular setback position using IVRO and augmentation genioplasty. The 3D orthognathic surgery including augmentation genioplasty simulation was performed. An excessively elongated proximal segment was sectioned following IVRO. The inferior part of the sectioned proximal bone fragment of the mandible was positioned to align with the requirements of advancement genioplasty. After ensuring that the placement of the fragment matched that of the simulated surgery, each bone fragment was fixed. At 1.5 years post-surgery, the grafted bone on the augmentation genioplasty was well maintained, with slight bone resorption. CONCLUSIONS: Augmentation genioplasty using the proximal bone fragment of the mandible, which is typically discarded in IVRO, reduces the surgical complications associated with chin osteotomy. When a secondary genioplasty is required, genioplasty with osteotomy, movement of the cut bone fragments, partial bone-shaving osteotomy, and additional bone grafting are viable options.

Cell Surface Modification-Mediated Primary Intestinal Epithelial Cell Culture Platforms for Assessing Host-Microbiota Interactions.

Background: Intestinal epithelial cells (IECs) play a crucial role in regulating the symbiotic relationship between the host and the gut microbiota, thereby allowing them to modulate barrier function, mucus production, and aberrant inflammation. Despite their importance, establishing an effective ex vivo culture method for supporting the prolonged survival and function of primary IECs remains challenging. Here, we aim to develop a novel strategy to support the long-term survival and function of primary IECs in response to gut microbiota by employing mild reduction of disulfides on the IEC surface proteins with tris(2-carboxyethyl)phosphine. Methods: Recognizing the crucial role of fibroblast-IEC crosstalk, we employed a cell surface modification strategy, establishing layer-to-layer contacts between fibroblasts and IECs. This involved combining negatively charged chondroitin sulfate on cell surfaces with a positively charged chitosan thin film between cells, enabling direct intercellular transfer. Validation included assessments of cell viability, efficiency of dye transfer, and IEC function upon lipopolysaccharide (LPS) treatment. Results: Our findings revealed that the layer-by-layer co-culture platform effectively facilitates the transfer of small molecules through gap junctions, providing vital support for the viability and function of primary IECs from both the small intestine and colon for up to 5 days, as evident by the expression of E-cadherin and Villin. Upon LPS treatment, these IECs exhibited a down-regulation of Villin and tight junction genes, such as E-cadherin and Zonula Occludens-1, when compared to their nontreated counterparts. Furthermore, the transcription level of Lysozyme exhibited an increase, while Mucin 2 showed a decrease in response to LPS, indicating responsiveness to bacterial molecules. Conclusions: Our study provides a layer-by-layer-based co-culture platform to support the prolonged survival of primary IECs and their features, which is important for understanding IEC function in response to the gut microbiota.

CLCC1 promotes membrane fusion during herpesvirus nuclear egress.

Herpesvirales are an ancient viral order that infects species from mollusks to humans for life. During infection, these viruses translocate their large capsids from the nucleus to the cytoplasm independently from the canonical route through the nuclear pore. Instead, capsids dock at the inner nuclear membrane and bud into the perinuclear space. These perinuclear enveloped virions fuse with the outer nuclear membrane releasing the capsids into the cytoplasm for maturation into infectious virions. The budding stage is mediated by virally encoded proteins. But the mediator of the subsequent fusion stage is unknown. Here, using a whole-genome CRISPR screen with herpes simplex virus 1, we identified CLCC1 as an essential host factor for the fusion stage of nuclear egress. Loss of CLCC1 results in a defect in nuclear egress, accumulation of capsid-containing perinuclear vesicles, and a drop in viral titers. In uninfected cells, loss of CLCC1 causes a defect in nuclear pore complex insertion. Viral homologs of CLCC1 are present in herpesviruses that infect mollusks and fish. Our findings uncover an ancient cellular membrane fusion mechanism important for the fundamental cellular process of nuclear envelope morphogenesis that herpesviruses hijack for capsid transport.

Korea4K: whole genome sequences of 4,157 Koreans with 107 phenotypes derived from extensive health check-ups.

BACKGROUND: Phenome-wide association studies (PheWASs) have been conducted on Asian populations, including Koreans, but many were based on chip or exome genotyping data. Such studies have limitations regarding whole genome-wide association analysis, making it crucial to have genome-to-phenome association information with the largest possible whole genome and matched phenome data to conduct further population-genome studies and develop health care services based on population genomics. RESULTS: Here, we present 4,157 whole genome sequences (Korea4K) coupled with 107 health check-up parameters as the largest genomic resource of the Korean Genome Project. It encompasses most of the variants with allele frequency >0.001 in Koreans, indicating that it sufficiently covered most of the common and rare genetic variants with commonly measured phenotypes for Koreans. Korea4K provides 45,537,252 variants, and half of them were not present in Korea1K (1,094 samples). We also identified 1,356 new genotype-phenotype associations that were not found by the Korea1K dataset. Phenomics analyses further revealed 24 significant genetic correlations, 14 pleiotropic associations, and 127 causal relationships based on Mendelian randomization among 37 traits. In addition, the Korea4K imputation reference panel, the largest Korean variants reference to date, showed a superior imputation performance to Korea1K across all allele frequency categories. CONCLUSIONS: Collectively, Korea4K provides not only the largest Korean genome data but also corresponding health check-up parameters and novel genome-phenome associations. The large-scale pathological whole genome-wide omics data will become a powerful set for genome-phenome level association studies to discover causal markers for the prediction and diagnosis of health conditions in future studies.

Observation of a new type of self-generated current in magnetized plasmas.

A tokamak, a torus-shaped nuclear fusion device, needs an electric current in the plasma to produce magnetic field in the poloidal direction for confining fusion plasmas. Plasma current is conventionally generated by electromagnetic induction. However, for a steady-state fusion reactor, minimizing the inductive current is essential to extend the tokamak operating duration. Several non-inductive current drive schemes have been developed for steady-state operations such as radio-frequency waves and neutral beams. However, commercial reactors require minimal use of these external sources to maximize the fusion gain, Q, the ratio of the fusion power to the external power. Apart from these external current drives, a self-generated current, so-called bootstrap current, was predicted theoretically and demonstrated experimentally. Here, we reveal another self-generated current that can exist in a tokamak and this has not yet been discussed by present theories. We report conclusive experimental evidence of this self-generated current observed in the KSTAR tokamak.

Development of Advanced Chimeric Endolysin to Control Multidrug-Resistant Staphylococcus aureus through Domain Shuffling.

The increase in the prevalence of multidrug-resistant (MDR) Staphylococcus aureus with strong biofilm-forming capacity poses a serious public health concern. Endolysins derived from bacteriophages are a promising solution for antibiotic resistance problems. However, some natural staphylococcal endolysins have several shortcomings, such as low solubility and high sequence homology among domains. To overcome these limitations, we constructed a hybrid endolysin library by swapping an enzymatically active domain (EAD) and a cell wall binding domain (CBD) of 12 natural staphylococcal endolysins. We found a novel chimeric endolysin, ClyC, which showed enhanced lytic activity against S. aureus compared to its parental endolysin forms. ClyC also exhibited strong antibacterial activity against S. aureus in various biomatrices, such as milk and blood. Moreover, the treatment of chimeric endolysin effectively eradicated biofilms of multidrug-resistant bacteria, including methicillin-resistant S. aureus (MRSA), S. epidermidis (MRSE), and S. aureus clinical isolates. In an in vivo mouse infection model, ClyC showed effective protection capability against methicillin-resistant Staphylococcus aureus (MRSA) without any toxic effects. Taken together, our data suggest that the chimeric endolysin ClyC can be considered a potential antibacterial agent against multidrug-resistant S. aureus and may have clinical relevance.

Structure and Function of the Autolysin SagA in the Type IV Secretion System of Brucella abortus.

A recent genetic study with Brucella abortus revealed the secretion activator gene A (SagA) as an autolysin component creating pores in the peptidoglycan (PGN) layer for the type IV secretion system (T4SS) and peptidoglycan hydrolase inhibitor A (PhiA) as an inhibitor of SagA. In this study, we determined the crystal structures of both SagA and PhiA. Notably, the SagA structure contained a PGN fragment in a space between the N- and C-terminal domains, showing the substrate-dependent hinge motion of the domains. The purified SagA fully hydrolyzed the meso-diaminopimelic acid (DAP)-type PGN, showing a higher activity than hen egg-white lysozyme. The PhiA protein exhibiting tetrameric assembly failed to inhibit SagA activity in our experiments. Our findings provide implications for the molecular basis of the SagA-PhiA system of B. abortus. The development of inhibitors of SagA would further contribute to controlling brucellosis by attenuating the function of T4SS, the major virulence factor of Brucella.

Anti-fibrotic activity of a rho-kinase inhibitor restores outflow function and intraocular pressure homeostasis.

Glucocorticoids are widely used as an ophthalmic medication. A common, sight-threatening adverse event of glucocorticoid usage is ocular hypertension, caused by dysfunction of the conventional outflow pathway. We report that netarsudil, a rho-kinase inhibitor, decreased glucocorticoid-induced ocular hypertension in patients whose intraocular pressures were poorly controlled by standard medications. Mechanistic studies in our established mouse model of glucocorticoid-induced ocular hypertension show that netarsudil both prevented and reduced intraocular pressure elevation. Further, netarsudil attenuated characteristic steroid-induced pathologies as assessed by quantification of outflow function and tissue stiffness, and morphological and immunohistochemical indicators of tissue fibrosis. Thus, rho-kinase inhibitors act directly on conventional outflow cells to prevent or attenuate fibrotic disease processes in glucocorticoid-induced ocular hypertension in an immune-privileged environment. Moreover, these data motivate the need for a randomized prospective clinical study to determine whether netarsudil is indeed superior to first-line anti-glaucoma drugs in lowering steroid-induced ocular hypertension.

In vivo estimation of optic nerve sheath stiffness using noninvasive MRI measurements and finite element modeling.

The optic nerve sheath (ONS) is biomechanically important. It is acted on by tension due to ocular movements, and by fluid shifts and/or alterations in intracranial pressure (ICP) in human disease, specifically in pathologies leading to intracranial hypertension. It has also been hypothesized that the ONS is acted on by altered ICP in astronauts exposed chronically to microgravity. However, a non-invasive method to quantify ONS biomechanical properties is not presently available; knowledge of such properties is desirable to allow characterization of the biomechanical forces exerted on the optic nerve head and other ocular structures due to the ONS. Thus, the primary objective of this study was to characterize the biomechanical properties (stiffness) of the human ONS in vivo as a necessary step towards investigating the role of ICP in various conditions, including Spaceflight Associated Neuro-ocular Syndrome (SANS). We acquired non-invasive magnetic resonance imaging (MRI) scans of ostensibly healthy subjects (n = 18, age = 30.4 ± 11.6 [mean ± SD] years) during supine and 15-degree head-down-tilt (HDT) postures, and extracted ONS contours from these scans. We then used finite element modeling to quantify ONS expansion due to postural changes and an inverse approach to estimate ONS stiffness. Using this non-invasive procedure, we estimated an in vivo ONS stiffness of 39.2 ± 21.9 kPa (mean ± SD), although a small subset of individuals had very stiff ONS that precluded accurate estimates of their stiffness values. ONS stiffness was not correlated with age and was higher in males than females.

A coupled reaction-diffusion-strain model predicts cranial vault formation in development and disease.

How cells utilize instructions provided by genes and integrate mechanical forces generated by tissue growth to produce morphology is a fundamental question of biology. Dermal bones of the vertebrate cranial vault are formed through the direct differentiation of mesenchymal cells on the neural surface into osteoblasts through intramembranous ossification. Here we join a self-organizing Turing mechanism, computational biomechanics, and experimental data to produce a 3D representative model of the growing cerebral surface, cranial vault bones, and sutures. We show how changes in single parameters regulating signaling during osteoblast differentiation and bone formation may explain cranial vault shape variation in craniofacial disorders. A key result is that toggling a parameter in our model results in closure of a cranial vault suture, an event that occurred during evolution of the cranial vault and that occurs in craniofacial disorders. Our approach provides an initial and important step toward integrating biomechanics into the genotype phenotype map to explain the production of variation in head morphology by developmental mechanisms.

Development of crash modification factors of horizontal curve design features for single-motorcycle crashes on rural two-lane highways: A matched case-control study.

Single-motorcycle crashes are overrepresented on horizontally curved segments of rural, two-lane, undivided (RTU) highways. However, the relationship between single-motorcycle crash risk and the design features of horizontal curves on RTU highways is not well-studied in existing literature. This study aims to quantify the effect of horizontal curve type and radius on the risk of single-motorcycle crashes with a matched case-control study that can address the issues of the low sample mean, aggregation bias, and uncontrolled confounders existing in the traditional cross-sectional study. In the matched case-control study, three matching factors-year, annual average daily traffic (AADT), and segment length-were selected to match controls (RTU segments without crash records) with cases (RTU segments with crash records). A total of 1601 cases and 16,010 matched controls over 11 years (2005-2015) were identified as matched-strata. A conditional logistic model was fitted on the matched-strata data to estimate the crash modification factors (CMFs) of horizontal curve design features for single-motorcycle crashes. The modeling results highlighted the interaction effects between curve type and radius on the risk of single-motorcycle crashes. Sharp (radius ≤ 1500 ft) non-reverse curves were identified as the riskiest curve design for motorcyclists, followed by sharp reverse curves and moderate (1500 ft < radius ≤ 3000 ft) reverse curves. The study also revealed that motorcyclists might take safety-compensation behaviors on sharp curves, narrow shoulders, and poor pavement conditions. Engineering and education countermeasures are suggested for comprehending curve presence and associated risk level, reducing curve entry speed, and improving safety awareness. Finally, the limitations of the study and possible solutions are discussed.

An evaluation of buccal infiltrations and inferior alveolar nerve blocks in pulpal anesthesia for mandibular first molars.

We compared the anesthetic efficacy of inferior alveolar nerve blocks (IANBs) with that of buccal infiltrations (BIs) in mandibular first molars. Using a crossover design, all subjects received a standard IANB or a BI of 1.7 mL of 4% articaine with 1:100,000 adrenaline (Septanest; Septodont, Saint-Maru-des-Fosses, France) on two appointments separated by at least 1 week. Pulpal anesthesia was determined by using an electric pulp tester. Electric pulp testing was repeated at 5, 8, 11, 15, 20, 25, and 30 minutes after the injections. Anesthesia was considered successful if the subject did not respond to the maximum output of the pulp tester at two or more consecutive time points. Fifty-four percent of the BI and 43% of the IANB were successful; the difference was not significant (p = 0.34). The onset of pulpal anesthesia was significantly faster with BI (p = 0.03). In conclusion, BI with 4% articaine for mandibular first molars can be a useful alternative for clinicians because compared with IANB it has a faster onset and a similar success rate.

A COMPUTATIONAL ANALYSIS OF BONE FORMATION IN THE CRANIAL VAULT USING A COUPLED REACTION-DIFFUSION-STRAIN MODEL.

Bones of the murine cranial vault are formed by differentiation of mesenchymal cells into osteoblasts, a process that is primarily understood to be controlled by a cascade of reactions between extracellular molecules and cells. We assume that the process can be modeled using Turing's reaction-diffusion equations, a mathematical model describing the pattern formation controlled by two interacting molecules (activator and inhibitor). In addition to the processes modeled by reaction-diffusion equations, we hypothesize that mechanical stimuli of the cells due to growth of the underlying brain contribute significantly to the process of cell differentiation in cranial vault development. Structural analysis of the surface of the brain was conducted to explore the effects of the mechanical strain on bone formation. We propose a mechanobiological model for the formation of cranial vault bones by coupling the reaction-diffusion model with structural mechanics. The mathematical formulation was solved using the finite volume method. The computational domain and model parameters are determined using a large collection of experimental data that provide precise three dimensional (3D) measures of murine cranial geometry and cranial vault bone formation for specific embryonic time points. The results of this study suggest that mechanical strain contributes information to specific aspects of bone formation. Our mechanobiological model predicts some key features of cranial vault bone formation that were verified by experimental observations including the relative location of ossification centers of individual vault bones, the pattern of cranial vault bone growth over time, and the position of cranial vault sutures.

A randomized controlled trial of various MTA materials for partial pulpotomy in permanent teeth.

OBJECTIVES: The aim of this study was to evaluate and compare the clinical applicability of various MTA materials as partial pulpotomy materials in permanent teeth. METHODS: Partial pulpotomy was performed on 104 permanent teeth from 82 people (mean 29.3±14.8years old), who met the inclusion criteria in randomized clinical trial. The teeth were divided into three groups: ProRoot MTA (n=33), OrthoMTA (n=36), RetroMTA (n=35). Clinical examination and radiographic comparison were carried out at 1, 3, 6 and 12 months after the treatment. Survival analysis was performed using the Kaplan-Meier survival curves and log rank tests. RESULTS: Partial pulpotomy sustained a high success rate up to 1year with no significant differences in the outcomes treated with three MTA materials: ProRoot MTA, 96.0%; OrthoMTA, 92.8%; RetroMTA, 96.0%. The Kaplan-Meier survival function curves showed no significant differences among three groups concerning clinical and radiographic cumulative survival rates. In addition, no potential prognostic factors related to the success rate of partial pulpotomy among age, sex, tooth type, root apex status, the site and type of pulp exposure, and the type of restoration were observed in log rank analysis. CONCLUSIONS: Partial pulpotomy with ProRoot MTA, OrthoMTA and RetroMTA had favorable results and clinical and radiographic results were not significantly different in three groups after 1year.

Effect of Topical Anesthesia on Pain from Needle Insertion and Injection and Its Relationship with Anxiety in Patients Awaiting Apical Surgery: A Randomized Double-blind Clinical Trial.

INTRODUCTION: We aimed to simultaneously investigate the effects of topical anesthesia on needle insertion and injection pain in the labial mucosa of the maxillary central incisors of patients awaiting apical surgery and to assess the relationship between patients' anxiety and pain scores. METHODS: Forty-four patients scheduled for apical surgery of the maxillary anterior incisor or canine were included, and all completed the Modified Dental Anxiety Scale (MDAS) questionnaire. One piece of Xylocaine (AstraZeneca, Sodertalje, Sweden) gauze was applied to the right or left side of the labial vestibule below the central incisor according to a randomization process, and 1 piece of water gauze was applied to the contralateral side of the labial vestibule. Each piece of gauze remained in place for 2 minutes. The subjects were asked to rate their pain according to the numeric rating scale immediately after needle insertion and anesthetic solution injection. RESULTS: Topical anesthetic application significantly reduced both insertion- and injection-related pain. Injection pain was significantly higher than insertion pain throughout the experiment. The difference in pain scores between the placebo and topical anesthetic groups was significantly greater for insertion pain than injection pain. The group with higher MDAS scores showed significantly higher pain scores, except for insertion pain reported by the topical anesthetic group, which did not show a significant difference between MDAS score groups. CONCLUSIONS: The topical anesthetic was highly effective for both insertion and injection pain during infiltration anesthesia in the maxillary central incisors. Highly anxious patients reported higher pain scores; however, topical anesthetics reduced the effect of anxiety on increasing pain.