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Background: Infected dentinal tubules are a possible source of bacteria that are responsible for the failure of root canal treatment. Therefore, disinfection of dentinal tubules by increasing the penetration of the irrigation solution is important for success in retreatment cases. This study utilized confocal laser scanning microscopy (CLSM) to assess and compare the impact of XPR, ultrasonic irrigation (UI) and sonic activation (SA) on NaOCl penetration into dentinal tubules following endodontic retreatment., Methods: A total of forty mandibular premolars were enrolled in this investigation. Following root canal preparation up to ProTaper X3 file (30/0.07), root canals were obturated with gutta-percha and bioceramic root canal sealer with single cone technique. The root canal filling materials were removed using ProTaper nickel-titanium rotary retreatment files until the working length was reached. The retreatment procedure was finalized using the ProTaper Next X4 (40/0.06). The teeth were divided into four groups based on the irrigation activation technique: control (conventional needle irrigation), SA, UI and XPR. During the final irrigation procedure, Rhodamine B dye was introduced to 5% NaOCl for visualization via CLSM. Subsequent to image acquisition, the maximum penetration, penetration percentage, and penetration area were calculated. Data were statistically analyzed using the Kruskal-Wallis, Friedman, and Bonferroni Dunn multiple comparison tests through R software (p < 0.05)., Results: In the middle third, UI yielded a significantly higher penetration percentage than the control group (p < 0.05). The UI and XPR groups showed increased penetration percentages in the coronal and middle thirds compared with the apical third (P < 0.05). Maximum penetration was notably reduced in the apical third than in comparison with the coronal and middle thirds in all groups (p < 0.05). In the control, SA and XP groups, the penetration area was ranked in descending order as coronal, middle and apical (p < 0.05). Conversely, in the ultrasonic group, the penetration area was significantly lower in the apical third than in the middle and coronal thirds (p < 0.05)., Conclusions: UI enhanced the penetration percentage in the middle third of the root compared with that in the control group. XPR and SA showed no significant effect on NaOCl penetration following retreatment., (© 2024. The Author(s).)

Objective: Microsporidial stromal keratitis (MSK) is an uncommon disease. Only several case series have been reported. We aimed to describe the clinical manifestations, histopathology and treatment outcomes of MSK., Methods and Analysis: Retrospective data of MSK diagnosed between January 2009 and December 2020 at the King Chulalongkorn Memorial Hospital, Bangkok, Thailand were retrieved. The diagnosis was made based on corneal scraping, corneal biopsy and corneal button histopathology findings. Detailed clinical characteristics, histopathological findings and treatment outcomes were reviewed and analysed., Results: 21 patients with MSK with a mean age of 63.8 years (SD 12.2) had an indolent disease onset with a median of 9 months (IQR 2.2-12.0). Five patients (23.8%) experienced ocular traumas. Herpes stromal keratitis was the most common preliminary diagnosis (33.3%), followed by non-specific ulcers and fungal keratitis. The most common corneal finding was multifocal grey-white lesions with anterior to mid-stromal infiltration and fluffy borders (66.7%). Pathogens were identified by modified trichrome staining of corneal scrapings in 11 of 14 cases (78.6%). Histopathological examination showed positive Ziehl-Neelsen staining in 17 of 19 cases (89.5%). All patients received surgical treatment, with 18 receiving therapeutic penetrating keratoplasty (TPK), 2 undergoing deep anterior lamellar keratoplasty and 1 undergoing femtosecond laser-assisted anterior lamellar keratoplasty. The overall cure rate was 76.2% after the first surgery and 95.2% after the second surgery., Conclusion: MSK can be easily underdiagnosed. Clues to diagnosis included a history of chronic refractory stromal infiltration and typical corneal findings of deep stromal infiltration, without epithelial defects. TPK is the preferred treatment for MSK., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The primary aim of this study was to investigate the impact of treatment with low-temperature plasma (LTP) for varying exposure durations on a multispecies cariogenic biofilm comprising C. albicans, L. casei, and S. mutans, as well as on single-species biofilms of L. casei and C. albicans, cultured on hydroxyapatite discs. Biofilms were treated with LTP-argon at a 10 mm distance for 30 s, 60 s, and 120 s. Chlorhexidine solution (0.12%) and NaCl (0.89%) were used as positive (PC) and negative controls (NC), respectively. Argon flow only was also used as gas flow control (F). Colony-forming units (CFU) recovery and confocal laser scanning microscopy (CLSM) were used to analyze biofilm viability. LTP starting at 30 s of application significantly reduced the viability of multispecies biofilms by more than 2 log 10 in all treated samples (p < 0.0001). For single-species biofilms, L. casei showed a significant reduction compared to PC and NC of over 1 log 10 at all exposure times (p < 0.0001). In the case of C. albicans biofilms, LTP treatment compared to PC and NC resulted in a significant decrease in bacterial counts when applied for 60 and 120 s (1.55 and 1.90 log10 CFU/mL, respectively) (p < 0.0001). A significant effect (p ≤ 0.05) of LTP in single-species biofilms was observed to start at 60 s of LTP application compared to F, suggesting a time-dependent effect of LTP for the single-species biofilms of C. albicans and L. casei. LTP is a potential mechanism in treating dental caries by being an effective anti-biofilm therapy of both single and multispecies cariogenic biofilms., (© 2024. The Author(s).)

Background: Epidemiological and toxicological studies indicate that increased exposure to air pollutants can lead to neurodegenerative diseases. To further confirm this relationship, we evaluated the association between exposure to ambient air pollutants and corneal nerve measures as a surrogate for neurodegeneration, using corneal confocal microscopy., Methods: We used population-based observational cross-sectional data from The Maastricht Study including N = 3635 participants (mean age 59.3 years, 51.6% were women, and 19.9% had type 2 diabetes) living in the Maastricht area. Using the Geoscience and hEalth Cohort COnsortium (GECCO) data we linked the yearly average exposure levels of ambient air pollutants at home address-level [particulate matter with diameters of ≤ 2.5 µm (PM2.5), and ≤ 10.0 µm (PM10), nitrogen dioxide (NO2), and elemental carbon (EC)]. We used linear regression analysis to study the associations between Z-score for ambient air pollutants concentrations (PM 2.5 , PM 10 , NO 2 , and EC) and Z-score for individual corneal nerve measures (corneal nerve bifurcation density, corneal nerve density, corneal nerve length, and fractal dimension)., Results: After adjustment for potential confounders (age, sex, level of education, glucose metabolism status, corneal confocal microscopy lag time, inclusion year of participants, smoking status, and alcohol consumption), higher Z-scores for PM 2.5 and PM 10 were associated with lower Z-scores for corneal nerve bifurcation density, nerve density, nerve length, and nerve fractal dimension [stβ (95% CI): PM 2.5 -0.10 (-0.14; -0.05), -0.04 (-0.09; 0.01), -0.11 (-0.16; -0.06), -0.20 (-0.24; -0.15); and PM 10 -0.08 (-0.13; -0.03), -0.04 (-0.09; 0.01), -0.08 (-0.13; -0.04), -0.17 (-0.21; -0.12)], respectively. No associations were found between NO 2 and EC and corneal nerve measures., Conclusions: Our population-based study demonstrated that exposure to higher levels of PM 2.5 and PM 10 are associated with higher levels of corneal neurodegeneration, estimated from lower corneal nerve measures. Our results suggest that air pollution may be a determinant for neurodegeneration assessed in the cornea and may impact the ocular surface health as well., (© 2024. The Author(s).)

Genomic information must be faithfully transmitted into two daughter cells during mitosis. To ensure the transmission process, interphase chromatin is further condensed into mitotic chromosomes. Although protein factors like condensins and topoisomerase IIα are involved in the assembly of mitotic chromosomes, the physical bases of the condensation process remain unclear. Depletion attraction/macromolecular crowding, an effective attractive force that arises between large structures in crowded environments around chromosomes, may contribute to the condensation process. To approach this issue, we investigated the "chromosome milieu" during mitosis of living human cells using an orientation-independent-differential interference contrast module combined with a confocal laser scanning microscope, which is capable of precisely mapping optical path differences and estimating molecular densities. We found that the molecular density surrounding chromosomes increased with the progression from prophase to anaphase, concurring with chromosome condensation. However, the molecular density went down in telophase, when chromosome decondensation began. Changes in the molecular density around chromosomes by hypotonic or hypertonic treatment consistently altered the condensation levels of chromosomes. In vitro, native chromatin was converted into liquid droplets of chromatin in the presence of cations and a macromolecular crowder. Additional crowder made the chromatin droplets stiffer and more solid-like. These results suggest that a transient rise in depletion attraction, likely triggered by the relocation of macromolecules (proteins, RNAs, and others) via nuclear envelope breakdown and by a subsequent decrease in cell volumes, contributes to mitotic chromosome condensation, shedding light on a different aspect of the condensation mechanism in living human cells., Competing Interests: Competing interests statement:The authors declare no competing interest.

New photostable and bright supramolecular complexes based on cucurbit[7]uril (CB7) host and diketopyrrolopyrole (DPP) guest dyes having two positively charged 4-(trimethylammonio)phenyl groups were prepared and characterized. The dye core displays large Stokes shift (in H 2 O, abs./emission max. 480/550 nm; ϵ~19 000, τ fl >4 ns), strong binding with the host (~560 nM K d ) and a linker affording fluorescence detection of bioconjugates with antibody and nanobody. Combination of protein-functionalized DPP dye with CB7 improves photostability and affords up to 12-fold emission gain. Two-color confocal and stimulated emission depletion (STED) microscopy with 595 nm or 655 nm STED depletion lasers shows that the presence of CB7 not only leads to improved brightness and image quality, but also results in DPP becoming cell-permeable., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Cardiac involvement in systemic light chain amyloidosis (AL) leads to chronic heart failure and is a major prognosis factor. Severe cellular defects are provoked in cardiac cells by tissue-deposited amyloid fibrils of misfolded free immunoglobulin light chains (LCs) and their prefibrillar oligomeric precursors., Objective: Understanding the molecular mechanisms behind cardiac cell cytotoxicity is necessary to progress in therapy and to improve patient management. One key question is how extracellularly deposited molecules exert their toxic action inside cardiac cells. Here we searched for direct evidence of amyloid LC uptake by cardiomyocytes in patient biopsies., Methods: We immunolocalized LCs in cardiac biopsies from four AL cardiac amyloidosis patients and analysed histopathological images by high resolution confocal microscopy and 3D image reconstruction., Results: We show, for the first time directly in patient tissue, the presence of LCs inside cardiomyocytes, and report their proximity to nuclei and to caveolin-3-rich areas. Our observations point to macropinocytosis as a probable mechanism of LC uptake., Conclusions: Internalisation of LCs occurs in patient cardiomyocytes. This event could have important consequences for the pathogenesis of the cardiac disease by enabling interactions between amyloid molecules and cellular organelles inducing specific signalling pathways, and might bring new insight regarding treatment.

Objectives: The low durability of composite resin restorations can be attributed to the degradation of the resin dentin bonding interface. Owing to the presence of hydrophilic components in the adhesive, the integrity of the resin dentin bonding interface is easily compromised, which, in turn, leads to a reduction in bond strength. The hydrophilic nature of the adhesive leads to water sorption, phase separation, and leaching of the resin component. Therefore, hydrophobic adhesives could effectively be used to stabilize the integrity and durability of the resin dentin bonding interface., Methods: We synthesized a novel hydrophobic dentin adhesive by partially replacing bisphenol A glycidyl dimethacrylate (Bis-GMA) with a light-curable urushiol monomer. The properties of the produced adhesive, including the degree of conversion, viscosity, contact angle, water sorption/solubility, and mechanical strength, were comprehensively examined and compared to those of the commercially adhesive Adper Single Bond 2 as a positive control. The adhesive properties were determined using microtensile bond strength measurements, laser confocal microscopy, scanning electron microscopy observations, and nanoleakage tests. Finally, the novel adhesive was subjected to biocompatibility testing to determine its potential cytotoxicity., Results: At a light-curable urushiol content of 20 %, the synthesized adhesive exhibited high degrees of conversion and hydrophobicity, low cytotoxicity, good mechanical properties, and outstanding adhesive strength., Conclusions: The introduction of the light-curable urushiol into dentin adhesives can significantly enhance their hydrophobic, mechanical, and bonding properties, demonstrating potential to significantly improve restoration longevity., Clinical Significance: The integration of light-curable urushiol has endowed the experimental adhesives with several enhanced functionalities. These notable benefits underscore the suitability of this monomer for expanded applications in clinical practice., Competing Interests: Declaration of competing interest This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. All study participants provided informed consent, and the study design was approved by the appropriate ethics review board. We have read and understood your journal's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A 32-year-old contact lens-wearing man with recent travel history to the Caribbean was referred for a corneal infiltrate in the left eye that worsened following 1-week of steroid-antibiotic therapy. Corneal cultures were obtained and sent to our facility's clinical microbiology laboratory for analysis. Same-day in vivo confocal microscopy revealed fungal elements. Nucleic acid sequencing performed on the isolated determined it to be a member of the entomopathogenic genus Metarhizium. Over the course of 3 months, the patient's corneal infiltrate ultimately resolved following topical natamycin 5 % therapy. This is the first reported case to have originated in the Caribbean and to utilize in vivo confocal microscopy to aid diagnosis. Our case also supports previous reports of success with natamycin therapy in treatment of Metarhizium sp. keratitis., Competing Interests: Declaration of competing interest The authors do not have any relevant conflicts of interest to disclose, (Copyright © 2024. Published by Elsevier Inc.)

The purpose of this study was to evaluate the antibacterial effect of silver nanoparticles (AgNPs) against Enterococcus faecalis and compare it with different irrigation solutions. This study was performed using 64 dentin blocks. E. faecalis suspension was dispensed to each sample and incubated under anaerobic conditions at 37°C throughout 21 days. After the inoculation period, the following solutions were added to each group and kept for 5 min: Group 1, 5.25% sodium hypochlorite (NaOCl); Group 2, 2.5% NaOCl; Group 3, 1% NaOCl; Group 4, 2% chlorhexidine (CHX); Group 5, 200 ppm hypochlorous acid (HOCl); and Group 6, AgNPs. The samples of positive control were treated with sterile saline. Biofilm viability assay was performed using the LIVE/DEAD BacLight Bacterial Viability Kit. Samples were examined using confocal laser scanning microscopy, respectively. There was no significant difference between the 5.25% NaOCl, 2.5% NaOCl, and 1%NaOCl groups (p > .05). However, these groups showed statistically higher antibacterial activity than the 2% CHX, 200 ppm HOCl, and AgNP groups. Also, 2% CHX showed greater percentage of dead cells compared with the AgNP and HOCl groups. While AgNPs group showed lower dead cell rate than all NaOCl groups and 2% CHX, it caused higher dead cells than 200 ppm HOCl group. The 200 ppm HOCl group showed the lowest percentage of dead cells (p < .05) Although the antibacterial effect of AgNPs is not as high as NaOCl and CHX, it has considerable bactericidal activity against E. faecalis and can be improved by further studies. RESEARCH HIGHLIGHTS: New antimicrobial approaches for root canal irrigation. Antimicrobial effect of silver nanoparticles against E. faecalis. Elimination of the biofilm layer for the success of endodontic treatment., (© 2024 The Authors. Microscopy Research and Technique published by Wiley Periodicals LLC.)

The mango tree (Mangifera indica) produces nutrient-dense fruit that is consumed across the world. Interestingly, despite widespread consumption of its fruit and its categorization in the Anacardiaceae family, allergic reactions to the mango tree and its components are comparatively rare, sometimes manifesting as allergic contact dermatitis (ACD). Evaluation of ACD most commonly includes a thorough clinical assessment with diagnostic support from patch testing and histopathologic review following skin biopsy. In recent years, reflectance confocal microscopy (RCM) has shown promising potential to join the repertoire of diagnostic tools for ACD by enabling dynamic and high-resolution imaging of contact dermatitis in vivo. Herein, we present a unique case of mango sap-induced ACD imaged and diagnosed in real time via RCM.

Objectives: Picosecond lasers with a microlens array can cause laser-induced optical breakdown (LIOBS) and LIC (Intradermal laser-induced cavitation) within high-fluence areas. This study aimed to describe the clinical, reflectance confocal microscopy (RCM), histopathological findings, and the characteristics of vacuoles caused by LIOBS and LIC in individuals with skin types III and IV., Materials and Methods: This study was performed on six Chilean healthy volunteers, males and females, aged 35-65 years old with Fitzpatrick skin phototypes III-IV. The laser was applied in the inner proximal area of the nondominant arm. RCM evaluation was performed 24 h later; 48 h later, skin biopsies were performed on the laser-treated areas. Clinical, histological, and RCM findings were recorded., Results: Every individual developed a 10 mm 2 area of clinical erythema in the treated area. Under RCM, all six volunteers had hyporeflective spherical structures at the level of the epidermis, consistent with intraepidermal vacuoles. Histopathological evaluation revealed different sizes of vacuoles in both the epidermis and dermis., Conclusion: The LIOBS and LIC processes and the secondary production of vacuoles could be highly valuable for effective dermal remodeling treatment and aid in promoting the production of new collagen, elastic fibers, and growth factors that could improve skin texture. These structures were visible under RCM and histopathological evaluation., (© 2024 Wiley Periodicals LLC.)

Background: Avelumab is a human antibody that targets the programmed cell death ligand-1 (PD-L1) protein in cancer cells. Novel anticancer therapies for renal cell carcinoma (RCC) consider cluster of differentiation 15 (CD15) and interleukin 17 receptor A (IL-17RA) as potential targets. Notably, the expression of PD-L1, CD15 and IL-17RA is dependent on signal transducer and activator of transcription 3 (STAT3)., Objectives: The aim of the study was to investigate whether targeting PD-L1 with avelumab alters the expression levels of CD15 and IL-17RA, and to assess the STAT3-mediated regulation of CD15 and IL-17RA., Material and Methods: We applied immunocytochemistry (ICC) and confocal laser scanning (CLS) microscopy to assess the expression and localization of the immunotherapy targets in 3 renal cancer cell lines and 1 healthy renal cell line., Results: After treatment with 20 ng/mL avelumab, renal cancer cells showed a reduction in STAT3 expression. The expression of CD15 increased in cancer cells that exhibited a high level of IL-17RA, and the membrane signal of CD15 was reduced. In other renal cancer cell lines, the expression of CD15 decreased. Conversely, the level of IL-17RA changed only in healthy renal cells after treatment with avelumab, with no impact on renal cancer cells., Conclusions: Our study suggests that the targeting of PD-L1 with avelumab alters the expression of CD15 and IL-17RA, which play an important prognostic and therapeutic role in novel anticancer therapy.

The striped dolphin (Stenella coeruleoalba) is a medium-sized pelagic dolphin with a single external nasal opening (blowhole) located in the rostral and dorsal regions of the skull. The nasal cavity is divided into three sections: the olfactory, respiratory, and vestibular areas. The surface epithelium lining the regio vestibularis is the first tissue in the nose to be directly affected by environmental antigens. Cetaceans have a significant amount of mucosa-associated lymphoid tissue (MALT) located throughout their bodies. The lymphoid tissue found in the nasal mucosa is known as nose- or nasopharynx-associated lymphoid tissue (NALT). NALT has not yet been studied in dolphins, but it has been identified and documented in humans and laboratory rodents. This study utilized toll-like receptor 2 (TLR2), CD4, Langerin/CD207, and inducible nitric oxide synthase to characterize, for the first time, immune cells in the mucosal regio vestibularis of the S. coeruleoalba nasal cavity using confocal microscopy immunofluorescence techniques. The findings revealed scattered immune cells immunoreactive to the tested antibodies, present in both the epithelial tissue lining the nasal cavity vestibulum and the underlying connective tissue. This study enhances our comprehension of the immune system of cetaceans. RESEARCH HIGHLIGHTS: This study provides new insights into NALT in S. coeruleoalba. This research deepens the knowledge of the skin of cetaceans., (© 2024 Wiley Periodicals LLC.)

Purpose: Our objective was to develop a methodology for assessing the linear energy transfer (LET) and relative biological effectiveness (RBE) in clinical proton and helium ion beams using fluorescent nuclear track detectors (FNTDs)., Methods and Materials: FNTDs were exposed behind solid water to proton and helium ( 4 He) ion spread-out Bragg peaks. Detectors were imaged with a confocal microscope, and the LET spectra were derived from the fluorescence intensity. The track- and dose-averaged LET (LET F and LET D , respectively) were calculated from the LET spectra. LET measurements were used as input on RBE models to estimate the RBE. Human alveolar adenocarcinoma cells (A549) were exposed at the same positions as the FNTDs. The RBE was calculated from the resulting survival curves. All measurements were compared with Monte Carlo simulations., Results: For protons, average relative differences between measurements and simulations were 6% and 19% for LET F and LET D , respectively. For helium ions, the same differences were 11% for both quantities. The position of the experimental LET spectra primary peaks agreed with the simulations within 9% and 14% for protons and helium ions, respectively. For the RBE models using LET D as input, FNTD-based RBE values ranged from 1.02 ± 0.01 to 1.25 ± 0.04 and from 1.08 ± 0.09 to 2.68 ± 1.26 for protons and helium ions, respectively. The average relative differences between these values and simulations were 2% and 4%. For A549 cells, the RBE ranged from 1.05 ± 0.07 to 1.47 ± 0.09 and from 0.89 ± 0.06 to 3.28 ± 0.20 for protons and helium ions, respectively. Regarding the RBE-weighted dose (2.0 Gy at the spread-out Bragg peak), the differences between simulations and measurements were below 0.10 Gy., Conclusions: This study demonstrates for the first time that FNTDs can be used to perform direct LET measurements and to estimate the RBE in clinical proton and helium ion beams., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abstract: Keratomycosis is a serious corneal infection associated with high ocular morbidity that can lead to severe vision loss. It is estimated to affect more than 1 million patients annually, most commonly occurring in tropical climates, and represents a growing threat to patients worldwide. Despite aggressive medical management, fungal infections have a higher rate of perforation requiring surgical intervention compared with other infectious etiologies. Early diagnosis and appropriate treatment are keys to preserving vision and saving patients' eyes.Timely diagnosis of fungal keratitis helps minimize corneal damage and scarring and increases the likelihood of a favorable outcome. Studies have shown that correct identification of fungal infections is often delayed up to 2 to 3 weeks after initial presentation. This leads to incorrect or ineffective treatment for many patients. Diagnostic techniques explored in this study include corneal scrapings with staining and culture, visualization with in vivo confocal microscopy, molecular diagnostic techniques including polymerase chain reaction, and recently developed omics-based technologies.Treatment of fungal keratitis begins with topical antifungals. Medical management has been proven to be effective, but with limitations including poor drug penetration and low bioavailability. Cases that do not respond to topical therapy require more invasive and novel treatments to control the infection. We review the clinical trials that have shaped current practice patterns, with focus on the efficacy of topical natamycin as the primary therapy for filamentous fungal keratitis. We explore additional management strategies such as localized intrastromal and intracameral injections of antifungal medications, photodynamic therapy, and surgical intervention., Competing Interests: A. Arboleda is a co-inventor on US patent PCT/US2021/024683. C.N. Ta has no conflict of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Treated or coated sutures promise to prevent contamination of wounds., Purpose: The purpose of the study was to coat surgical sutures with a new quaternary ammonium silane (QAS) antimicrobial compound at two different application temperatures and then to evaluate the resulting structural, physical, mechanical, and biological properties., Study Design, Setting, Sample: In vitro and in vivo studies were conducted using male albino Wistar rats approved by the Joint Ethical Committee of IMU and Postgraduate Medical Institute, Lahore. Only suture samples, coated uniformly with verified presence of the compound and of adequate length were used. Samples which were not coated uniformly and with inadequate length or damaged were excluded., Predictor Variable: Predictor variables were sutures with and without QAS coatings and different temperatures. Sutures were coated with QAS at 0.5 and 1.0% wt/vol using the dip coating technique and sutures with and without QAS coating were tested at 25 and 40 °C temperatures., Main Outcome Variable(s): Outcome variables of structural and physico-mechanical properties of QAS-coated and non-coated sutures were measured using Fourier transform infrared spectroscopy (for structural changes), confocal laser and scanning electron (for diameter changes), and tensile strength/modulus (for mechanical testing). Biologic outcome variables were tested (bacterial viability); macrophage cultures from Wistar rats were tested (M1/M2 polarization detecting IL-6 and IL-10). Macrophage cells were analyzed with CD80+ (M1) and CD163+ (M2). Chemotaxis index was calculated as a ratio of quantitative fluorescence of cells., Covariates: Not applicable., Analyses: Ordinal data among groups were compared using the Wilcoxon Mann-Whitney U test along with the comparison of histological analysis using the Wilcoxon Sign-rank test (P < .05)., Results: Fourier transform infrared spectroscopy peak at 1490 cm -1 confirmed the presence of QAS on suture's surfaces with a significant increase (P < .05) in diameter (0.99 ± 0.5-mm) and weight (0.77 ± 0.02-mg) observed for 1% QAS groups treated at 40 °C. Non-coated samples heated at 25 °C had significantly (P < .05) less diameters (0.22 ± 0.03-mm) and weights (0.26 ± 0.06-mg). Highest tensile strength/modulus was observed for 0.5% QAS-coated samples which also had significantly higher antibacterial characteristics than other sutures (P < .05). QAS-coated sutures significantly increased M1 and M2 markers., Conclusion and Relevance: QAS coating conferred antibacterial action properties without compromising the physical and mechanical properties of the suture., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: To investigate the tear proteomic and neuromediator profiles, in vivo confocal microscopy (IVCM) imaging features, and clinical manifestations in neuropathic corneal pain (NCP) patients., Design: Cross-sectional study., Methods: A total of 20 NCP patients and 20 age-matched controls were recruited. All subjects were evaluated by corneal sensitivity, Schirmer test, tear break-up time, and corneal and ocular surface staining, Ocular Surface Disease Index and Ocular Pain Assessment Survey questionnaires were administered, as well as IVCM examinations for corneal nerves, microneruomas, and epithelial and dendritic cells. Tears were collected for neuromediator and proteomic analysis using enzyme-linked immunosorbent assay and data-independent acquisition mass spectrometry., Results: Burning and sensitivity to light were the 2 most common symptoms in NCP. A total of 188 significantly dysregulated proteins, such as elevated metallothionein-2, creatine kinases B-type, vesicle-associated membrane protein 2, neurofilament light polypeptide, and myelin basic protein, were identified in the NCP patients. The top 10 dysregulated biological pathways in NCP include neurotoxicity, axonal signaling, wound healing, neutrophil degradation, apoptosis, thrombin signaling mitochondrial dysfunction, and RHOGDI and P70S6K signaling pathways. Compared to controls, the NCP cohort presented with significantly decreased corneal sensitivity (P < .001), decreased corneal nerve fiber length (P = .003), corneal nerve fiber density (P = .006), and nerve fiber fractal dimension (P = .033), as well as increased corneal nerve fiber width (P = .002), increased length, total area and perimeter of microneuromas (P < .001, P < .001, P = .019), smaller corneal epithelial size (P = .017), and higher nerve growth factor level in tears (P = .006)., Conclusions: These clinical manifestations, imaging features, and molecular characterizations would contribute to the diagnostics and potential therapeutic targets for NCP., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ultrasound spectroscopy and confocal laser scanning microscopy (CLSM) methods were developed to visualize the interaction between sodium caseinate (SC) and whey protein isolate (WPI) with a mild preheat treatment (57°C, 10 min) followed by adding glucono-δ-lactone (GDL). Ultrasonic velocity changes during incubation at 25°C after adding GDL for four kinds of mixtures (no-treated SC plus no-treated WPI, preheated SC plus no-treated WPI, no-treated SC plus preheated WPI and preheated SC plus preheated WPI) were monitored. The results reveal that the mild preheating treatment of the proteins affected the timing of the increase in compressibility of each system. CLSM observation with individualized dyes which have different maxima of excitation and emission wavelengths, showed the preheated SC plus no-treated WPI mixture had a slightly coarse structure and the highest correlation coefficient, suggesting the highest colocalization of the SC and WPI among the four kinds of mixed-protein systems. Furthermore, the scanning electron microscopy (SEM) observation suggests that there are some differences among the gels, namely, preheated WPI leads to the formation of developed three-dimensional gel networks with filamentous structures, whereas SC promotes the formation of cluster-like crowded networks composed of more fine aggregated particles instead of developed filamentous structures. These results demonstrated that although SC is known as a heat-stable protein, pretreated SC could lead to an increase of the collaboration with WPI in the presence of GDL. This finding anticipated the possibility creating a food material with another texture using a milk-protein mixed system., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bacillus cereus is a well-known foodborne pathogen that can cause human diseases, including vomiting caused by emetic toxin, cereulide, requiring 10 5 -10 8 cells per gram to cause the disease. The bacterial cells may be eliminated during processing, but cereulide can survive in most processing techniques due to its resistance to high temperatures, extreme pH and proteolytic enzymes. Herein, we reported dynamic processes of biofilm formation of four different types and cereulide production within the biofilm. Confocal laser scanning microscopy (CLSM) images revealed that biofilms of the four different types reach each stage at different time points. Among the extracellular polymeric substances (EPS) components of the four biofilms formed by the emetic B. cereus F4810/72 strain, proteins account for the majority. In addition, there are significant differences (p < 0.05) in the EPS components at the same stage among biofilms of different types. The time point at which cereulide was first detected in the four types of biofilms was 24 h. In the biofilm of B. cereus formed in ultra-high-temperature (UHT) milk, the first peak of cereulide appeared at 72 h. The cereulide content of the biofilms formed in BHI was mostly higher than that of the biofilms formed in UHT milk. This study contributes to a better understanding of food safety issues in the industry caused by biofilm and cereulide toxin produced by B. cereus., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Significance: This study establishes an increased risk of developing dry eye disease (DED) in patients with diabetic peripheral neuropathy using validated diagnostic criteria for both conditions., Purpose: The disruption of ocular surface homeostasis has been associated with diabetes. However, it remains unclear if this association is independently influenced by peripheral neuropathy secondary to diabetes. This study aimed to investigate the clinical signs and symptoms of DED and their association with the severity of peripheral neuropathy in participants with type 2 diabetes., Methods: This prospective cross-sectional study recruited 63 participants with type 2 diabetes. All participants underwent a detailed assessment of DED using dry eye questionnaires (Ocular Surface Disease Index, Dry Eye Questionnaire-5), tear osmolarity, lipid layer thickness, noninvasive keratographic tear breakup time, phenol red thread test (PRT), and ocular surface staining. Corneal nerve morphology was imaged using corneal confocal microscopy. Based on the Total Neuropathy Scale, participants were stratified into no/mild (n = 48) and moderate/severe (n = 15) neuropathy groups., Results: Dry eye disease was diagnosed in 31 participants (50%) of the total cohort, and the odds of developing DED in the moderate/severe neuropathy group were four times (95% confidence interval, 1.10 to 13.80; p=0.030) higher compared with the no/mild neuropathy group. The Dry Eye Questionnaire-5 scores were significantly higher (p=0.020), and PRT values (p=0.048) and corneal nerve fiber length (p<0.001) were significantly reduced in the moderate/severe neuropathy group compared with the no/mild neuropathy group. In regression analysis, neuropathy scores were independently associated with PRT measurements ( β = -0.333, p=0.023) and nerve fiber length ( β = -0.219, p=0.012) while adjusting for age, gender, hemoglobin A 1c , and duration of diabetes., Conclusions: Type 2 diabetic patients with peripheral neuropathy have a risk of developing DED, which increases with the severity of neuropathy. The observation that worsening peripheral neuropathy is associated with reduced tear secretion suggests that it may contribute to aqueous insufficiency., Competing Interests: Conflict of Interest Disclosure: None of the authors have reported a financial conflict of interest., (Copyright © 2024 American Academy of Optometry.)

Characterizing the physiological response of bacterial cells to antibiotics is crucial for designing diagnostic techniques, treatment choices, and drug development. While bacterial cells at sublethal doses of antibiotics are commonly characterized, the impact of exposure to high concentrations of antibiotics on bacteria after long-term serial exposure and their effect on withdrawal need attention for further characterization. This study investigated the effect of increasing imipenem concentrations on carbapenem-susceptible (S) and carbapenem-resistant (R) E. coli on their growth adaptation and cell surface structure. We exposed the bacterial population to increasing imipenem concentrations through 30 exposure cycles. Cell morphology was observed using a 3D laser scanning confocal microscope (LSCM) and transmission electron microscope (TEM). Results showed that the exposure resulted in significant morphological changes in E. coli (S) cells, while minor changes were seen in E. coli (R) cells. The rod-shaped E. coli (S) gradually transformed into round shapes. Further, the exposed E. coli (S) cells' surface area-to-volume ratio (SA/V) was also significantly different from the control, which is non-exposed E. coli (S). Then, the exposed E. coli (S) cells were re-grown in antibiotic-free environment for 100 growth cycles to determine if the changes in cells were reversible. The results showed that their cell morphology remained round, showing that the cell morphology was not reversible. The morphological response of these cells to imipenem can assist in understanding the resistance mechanism in the context of diagnostics and antibacterial therapies., (© 2024. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

We aimed to evaluate the penetration of endodontic cement following the removal of calcium hydroxide (Ca(OH)2) dressing using the XP-endo Finisher in association with different irrigating solutions. Sixty premolars were instrumented and applied with a Ca(OH)2 dressing. To remove Ca(OH)2, the teeth were divided into six groups, each with a different volume of sodium hypochlorite (NaOCl) and ethylenediaminetetraacetic acid (EDTA), as well as solution stirring time with XP-endo Finisher (0, 30, and 60 sec). Root canals were filled using the lateral condensation technique. Fluorescein dye was added to the cement for microscopic laser scanning analysis. In the generated images, linear measurements were taken in micrometers, and their averages were calculated. To analyze the perimeter penetration ratio of the cement, the total perimeter of the canal and the segment of the total perimeter of the canal where the endodontic cement penetrated into the dentinal tubules were measured in micrometers. We found that using an XP-endo Finisher in irrigation was more effective than using a needle and syringe during the extension and penetration of endodontic cement. Shaking with XP-endo Finisher with 17% EDTA increased the extent and perimeter of the penetration of the endodontic cement into the dentinal tubules. However, using the XP-endo Finisher with EDTA only was more efficient than using the instrument interchangeably in NaOCl and EDTA. Although XP-endo Finisher contributes to the removal of Ca(OH)2, none of the protocols or instruments used removed all Ca(OH)2 from the root system. [ABSTRACT FROM AUTHOR]

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Aim: The aim of this study was to evaluate the effect of ethanol-conditioned dentin on endodontic sealer penetration into dentinal tubules by confocal laser scanning microscopy (CLSM). Methods: Forty human maxillary anterior teeth were instrumented and divided into four groups (n = 10) according to the drying methods: 1) wet: vacuum only, 2) paper points: vacuum + absorbent paper points, (3) 70% ethanol: 70% ethanol (1 min) + vacuum + absorbent paper points, and (4) 100% ethanol: 100% ethanol (1 min) + vacuum + absorbent paper points. All root canals were filled with resin-based endodontic sealer. Four sections from each third (cervical, middle, and apical) were examined by CLSM. Root canal wall perimeter infiltrated by sealer, maximum depth of sealer penetration, percentage of penetrated area, and fluorescence intensity of rhodamine B were measured. Statistical analysis was performed by analysis of variance and Tukey’s tests (α = 0.05). Results: No statistical difference was found when percentage of root canal wall coverage infiltrated by sealer were compared. The groups in which ethanol solutions were used presented greater depth of sealer penetration, higher percentage of penetrated area, and higher fluorescence intensity of rhodamine B (p< 0.05) when compared with the wet and paper point groups. Overall, 100% ethanol produced better results than 70% ethanol, except for rhodamine B intensity (cervical third). In addition, the absorbent paper points drying method behaved better than did vacuum only group, except for rhodamine B intensity (apical third). Conclusion: Ethanol-conditioned dentin improved the penetration of resin-based sealer into dentinal tubules, especially at the concentration of 100%.

Cystinosis is a rare autosomal recessive lysosomal storage disease associated with high mortality and morbidity rates. The most distinctive ocular manifestations of cystinosis are photophobia, tearing, and blurred vision. Herein, we assessed the ocular involvement of four patients from two families diagnosed with infantile nephropathic cystinosis using optical coherence tomography (OCT) and in vivo confocal microscopy (IVCM). Anterior segment OCT demonstrated multiple hyperreflective punctate deposits, and IVCM revealed needle-shaped bright crystal deposits in the corneal stroma in all patients. Three patients also had crystal deposits in the epithelium, where epithelial cell disruption was observed. Crystal deposits around the subepithelial nerve plexus were noted in some sections. In one patient, round and needle-shaped bright deposits along with inflammatory cells were observed in the limbal region of the conjunctiva. Infrared fundus images of two female siblings revealed hyperreflective crystal-like deposits around the optic disc, macula, and peripheral retina, and enhanced depth imaging OCT showed accumulation of crystals in all layers of the retina., Competing Interests: Conflict of Interest: No conflict of interest was declared by the authors., (©Copyright 2024 by the Turkish Ophthalmological Association / Turkish Journal of Ophthalmology published by Galenos Publishing House.)

Objectives: To evaluate the marginal integrity (MI%) and to characterize specific properties of a thermoviscous bulk-fill resin composite, two regular bulk-fill resin composites, and a non-bulk-fill resin composite., Materials and Methods: VisCalor bulk (VBF), Filtek One Bulk Fill (OBF), and Aura Bulk Fill (ABF) were evaluated. Filtek Z250 XT (ZXT) was used as non-bulk-fill control. MI% was evaluated in standardized cylindrical cavities restored with the composites by using a 3D laser confocal microscope. The following properties were characterized: volumetric polymerization shrinkage (VS%), polymerization shrinkage stress (Pss), degree of conversion (DC%), microhardness (KHN), flexural strength (FS), and elastic modulus (EM). Data were analyzed by one-way and two-way ANOVA, and Tukey HSD post-hoc test (α = 0.05)., Results: VBF presented the highest MI% and the lowest VS% and Pss (p < 0.05). DC% ranged from 59.4% (OBF) to 71.0% (ZXT). ZXT and VBF presented similar and highest KHN than OBF and ABF (p < 0.05). ABF presented the lowest FS (p < 0.05). EM ranged from 5.5 GPa to 7.7 GPa, with the values of ZXT and VBF being similar and statistically higher than those of OBF and ABF (p < 0.05)., Conclusions: Thermoviscous technology employed by VisCalor bulk was able to improve its mechanical behavior comparatively to regular bulk-fill resin composites and to contribute to a better marginal integrity in restorations built up in cylindrical cavities with similar geometry to a class I cavity as well. Although presenting overall better physicomechanical properties, Z250 XT presented the worst MI%., Clinical Relevance: The marginal integrity, which is pivotal for the success of resin composite restorations, could be improved using VisCalor bulk-fill. The worst MI% presented by Z250 XT reinforces that non-bulk-fill resin composites shall not be bulk-inserted in the cavity to be restored., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The nuclear pore complexes on the nuclear membrane serve as the exclusive gateway for communication between the nucleus and the cytoplasm, regulating the transport of various molecules, including nucleic acids and proteins. The present work investigates the kinetics of the transport of negatively charged graphene quantum dots through nuclear membranes, focusing on quantifying their transport characteristics. Experiments are carried out in permeabilized HeLa cells using time-lapse confocal fluorescence microscopy. Our findings indicate that negatively charged graphene quantum dots exhibit rapid transport to the nuclei, involving two distinct transport pathways in the translocation process. Complementary experiments on the nuclear import and export of graphene quantum dots validate the bi-directionality of transport, as evidenced by comparable transport rates. The study also shows that the negatively charged graphene quantum dots possess favorable retention properties, underscoring their potential as drug carriers., (© 2024. The Author(s).)

The aim of this study is to evaluate the effect of different irrigation activation methods on root canal sealer penetration in teeth with simulated internal root resorption (IRR) and calcium hydroxide (CH) applied using a confocal laser scanning microscope (CLSM). 60 incisors with a single root and a single canal were included in the study. IRR cavities were created in the middle third of the root canal, and CH was placed. The samples were randomly divided into 4 groups (n = 15) according to the irrigation activation method to be tested: standard needle irrigation (SNI), sonic activation (EDDY), photon-induced photoacoustic flow (PIPS), and shock wave enhanced emission photoacoustic flow (SWEEPS). After irrigation activation applications, the root canals were obturated. Sections of 1.0 ± 0.1 mm were taken from the apical, middle, and coronal regions of each sample. The penetration area (µm 2 ) and maximum penetration depth (µm) of the root canal sealer were examined by CLSM and analyzed using ImageJ software. Statistical analysis was performed with a one-way ANOVA and post-hoc Tukey test at the P < 0.05 significance level. Among all irrigation activation methods tested, both the penetration area and maximum penetration depth of the root canal sealer were greater in the coronal region than in the apical region (p < 0.05). In the IRR region, there was no difference in terms of maximum penetration depth between PIPS and SWEEPS (p > 0.05), it was highest in SWEEPS (p < 0.05). PIPS and SWEEPS were better than other irrigation activation methods in the penetration of root canal sealer in the resorption areas of teeth with IRR., (© 2024. The Author(s), under exclusive licence to Springer-Verlag London Ltd., part of Springer Nature.)

Objective: The red-complex bacteria Porphyromonas gingivalis and Tannerella forsythia together with Fusobacterium nucleatum are essential players in periodontitis. This study investigated the bacterial interplay with human periodontal ligament mesenchymal stromal cells (hPDL-MSCs) which act in the acute phase of periodontal infection., Design: The capability of the bacteria to induce an inflammatory response as well as their viability, cellular adhesion and invasion were analyzed upon mono- and co-infections of hPDL-MSCs to delineate potential synergistic or antagonistic effects. The expression level and concentration of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1 were measured using qRT-PCR and ELISA. Viability, invasion, and adhesion were determined quantitatively using agar plate culture and qualitatively by confocal microscopy., Results: Viability of P. gingivalis and T. forsythia but not F. nucleatum was preserved in the presence of hPDL-MSCs, even in an oxygenated environment. F. nucleatum significantly increased the expression and concentration of IL-6, IL-8 and MCP-1 in hPDL-MSCs, while T. forsythia and P. gingivalis caused only a minimal inflammatory response. Co-infections in different combinations had no effect on the inflammatory response. Moreover, P. gingivalis mitigated the increase in cytokine levels elicited by F. nucleatum. Both red-complex bacteria adhered to and invaded hPDL-MSCs in greater numbers than F. nucleatum, with only a minor effect of co-infections., Conclusions: Oral bacteria of different pathogenicity status interact differently with hPDL-MSCs. The data support P. gingivalis' capability to manipulate the inflammatory host response. Further research is necessary to obtain a comprehensive picture of the role of hPDL-MSCs in more complex oral biofilms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study evaluated the effect on adhesive layer thickness, bond strength, and adhesive failure pattern of the application of universal adhesive (Scotchbond Universal) using either manual or rotary brush in dentin previously impregnated with bioceramic sealer (Sealer Plus BC) using a manual brush, at 24 h and 1 year. Eighty-eight bovine crowns were divided into four groups (n = 22) according to the intervention: (i) use of bioceramic sealer and adhesive application using manual brush, (ii) use of bioceramic sealer and adhesive application using rotary brush, (iii) use of resin sealer and adhesive application using manual brush, and (iv) use of resin sealer and adhesive application using rotary brush. Subsequently, specimens were restored with a composite resin (Filtek Z-250). Adhesive layer thickness was evaluated using confocal microscopy. Bond strength was assessed using the microtensile bond strength test, and adhesive failure pattern was evaluated under a stereomicroscope. Data were analyzed using two-way ANOVA/Tukey tests. Specimens where a rotary brush had been used exhibited lower adhesive layer thickness. Specimens treated with resin sealer and using a manual brush showed lower bond strength values and a higher occurrence of adhesive failures at 24 h and 1 year than specimens treated with bioceramic sealer and using rotary brush for adhesive application., (© 2024 Scandinavian Division of the International Association for Dental Research. Published by John Wiley & Sons Ltd.)

Objectives: To evaluate the wear resistance of three additively manufactured dental crown materials (NextDent C&B MFH, Saremco print CROWNTEC and Bego VarseoSmile Crown) under two environmental conditions (dry and artificial saliva), two loads (49 N and 70 N) and two surface treatments (polished and glazed)., Methods: A total of 120 specimens were divided into 24 groups and tested for wear under two loads (49 N and 70 N), surface treatment (polished or glazed), and environment (dry or submerged in artificial saliva). All samples underwent reciprocating wear testing at 1 Hz using a wear simulator, replicating 48 months of In Vivo conditions with a stainless-steel ball as the antagonist. The coefficient of friction (CoF), surface roughness, volumetric and vertical wear loss were measured and statistically analysed. Confocal microscopy assessed the surface properties of crown materials and the antagonists., Results: The NextDent material demonstrated the most homogenous wear, with relatively low vertical and volumetric loss across all groups (p < 0.004). NextDent and Bego materials performed similarly in artificial saliva regardless of the load type (p > 1.000). The CoF remained below 0.3 for all groups. All groups exhibited significant increases in surface roughness after testing, however, this did not correlate with an increase in the CoF. Confocal analysis revealed material deformities due to load and notable scratch marks on the stainless-steel antagonists., Conclusion: It was found that all investigated addtively manufactured materials can be suggested for provisional use. Both vertical loss and volumetric loss results should be included for material evaluation. CoF and surface roughness should be implemented into wear evaluation., Clinical Significance: This study highlights the practical value of additively manufactured dental crown materials, particularly for provisional restorations. However, their extended use requires careful consideration of individual patient needs, emphasising the need for judicious clinical application evaluation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in the paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objective: Investigate the bioactivity and stability of Rhodiola rosea (RR) fractions as a natural source of prodelphinidin gallate (PDg) on dentin collagen via analysis of the viscoelastic and resin-dentin adhesive properties of the dentin matrix., Methods: The biomimicry and stability of RR subfractions (F1, F2, F3 and F4) with collagen were determined by dynamic mechanical analysis (DMA). DMA used a strain sweep method to assess the dentin matrix viscoelastic properties [storage (E'), loss (E"), and complex (E*) moduli and tan δ] after treatment, 7-, 30- and 90-days of storage in simulated body fluids (SBF). Resin-dentin interface properties were assessed after 1 and 90-days in SBF by microtensile bond strength test and confocal laser scanning microscopy. Data were analyzed using two and one-way ANOVA and post-hoc tests (α = 0.05)., Results: RR fractions increased dentin matrix complex (96 - 69 MPa) and storage (95 - 68 MPa) moduli, compared to the control (∼9 MPa) in the ranking order: F2 ≥ F3 = F1 = F4 > control (p < 0.001). Treatment did not affect tan δ values. After 30- and 90-days, RR-treated dentin E*, E' and tan δ decreased (p < 0.001). F2 fraction yielded the highest microtensile bond strength (43.9 MPa), compared to F1, F4 (35.9 - 31.7 MPa), and control (29 MPa). RR-treated interfaces mediated stable surface modifications and enhanced collagen-methacrylate resin interactions at the bioadhesive interface., Significance: Prodelphinidin gallates from RR are potent and reasonably stable biomimetic agents to dentin. Higher potency of F2 fraction with the dentin matrix and the adhesive interface is associated with a degree of polymerization of 2-3 and gallo(yl) motifs., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objectives: This study aims to evaluate antibacterial effects of silver diamine fluoride (SDF), SDF/potassium iodide (KI), and nanosilver fluoride (NSF)., Methods: Antimicrobial activity of sterile saline, 5% sodium hypochlorite (NaOCl), 2% chlorhexidine (CHX), SDF, SDF/KI, NSF, and KI solutions against Streptococcus mutans and Lactobacillus casei was assessed through disc diffusion tests. A dual-species biofilm of S. mutans-L. casei was formed on 48 enamel samples, divided into six groups (n = 8). Group 1 was treated with sterile saline, Group 2 with 5% NaOCl, Group 3 with 2% CHX, Group 4 with SDF, Group 5 with SDF/KI, and Group 6 with NSF. The samples were analysed using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Statistical analysis utilized Shapiro-Wilk and Kruskal-Wallis tests and multiple comparisons were conducted using Dunn test., Results: SDF, SDF/KI, and NaOCl displayed significantly higher antibacterial activity against dual-species biofilm compared to NSF and CHX (p < 0.050)., Conclusions: In conclusion, SDF and SDF/KI demonstrated greater antibacterial activity than NSF. SDF's antibacterial activity was unaffected by KI. Further research is needed to determine the appropriate content and concentration for achieving effective antibacterial activity with NSF., Clinical Significance: The use of silver-containing materials is increasing in popularity within pediatric dentistry. In this study, an endeavor has been made to assist pediatric dentists in determining which solution might be more advantageous for preventing caries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Aim: To evaluate the adhesion of mono and duospecies biofilm on a commercially available dental implant surface coated with hydroxyapatite nanoparticles (nanoHA)., Material and Methods: Titanium discs were divided into two groups: double acid-etched (AE) and AE coated with nanoHA (NanoHA). Surface characteristics evaluated were morphology, topography, and wettability. Mono and duospecies biofilms of Streptococcus sanguinis (S. sanguinis) and Candida albicans (C. albicans) were formed. Discs were exposed to fetal bovine serum (FBS) to form the pellicle. Biofilm was growth in RPMI1640 medium with 10% FBS and 10% BHI medium for 6 h. Microbial viability was evaluated using colony-forming unit and metabolic activity by a colorimetric assay of the tetrazolium salt XTT. Biofilm architecture and organization were evaluated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM)., Results: AE surface had more pores, while NanoHA had even nanoHA crystals distribution. Roughness was similar (AE: 0.59 ± 0.07 µm, NanoHA: 0.69 ± 0.18 µm), but wettability was different (AE: Θw= 81.79 ± 8.55°, NanoHA: Θw= 53.26 ± 11.86°; P = 0.01). NanoHA had lower S. sanguinis viability in monospecies biofilm (P = 0.007). Metabolic activity was similar among all biofilms. In SEM both surfaces on C. albicans biofilm show a similar distribution of hyphae in mono and duospecies biofilms. AE surface has more S. sanguinis than the NanoHA surface in the duospecies biofilm. CLSM showed a large proportion of live cells in all groups., Conclusions: The nanoHA surface reduced the adhesion of S. sanguinis biofilm but did not alter the adhesion of C. albicans or the biofilm formed by both species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Plant-pathogenic fungi produce toxins as virulence factors in many plant diseases. In Cercospora leaf blight of soybean caused by Cercospora cf. flagellaris , symptoms are a consequence of the production of a perylenequinone toxin, cercosporin, which is light-activated to produce damaging reactive oxygen species. Cercosporin is universally toxic to cells, except to the cells of the producer. The current model of self-resistance to cercosporin is largely attributed to the maintenance of cercosporin in a chemically reduced state inside hyphae, unassociated with cellular organelles. However, in another perylenequinone-producing fungus, Phaeosphaeria sp., the toxin was specifically sequestered inside lipid droplets (LDs) to prevent reactive oxygen species production. This study hypothesized that LD-based sequestration of cercosporin occurred in C . cf. flagellaris and that lipid-inhibiting fungicides could inhibit toxin production. Confocal microscopy using light-cultured C . cf. flagellaris indicated that 3-day-old hyphae contained two forms of cercosporin distributed in two types of hyphae. Reduced cercosporin was uniformly distributed in the cytoplasm of thick, primary hyphae, and, contrary to previous studies, active cercosporin was observed specifically in the LDs of thin, secondary hyphae. The production of hyphae of two different thicknesses, a characteristic of hemibiotrophic plant pathogens, has not been documented in C . cf. flagellaris . No correlation was observed between cercosporin production and total lipid extracted, and two lipid-inhibiting fungicides had little effect on fungal growth in growth-inhibition assays. This study lays a foundation for exploring the importance of pathogen lifestyle, toxin production, and LD content in the pathogenicity and symptomology of Cercospora ., Competing Interests: The author(s) declare no conflict of interest.

Introduction: There have been reports on the effects of epigallocatechin gallate (EGCG) against Streptococcus mutans viability and acidogenesis. However, the effects of EGCG on the virulence of S. mutans biofilm development have yet to be fully investigated using validated cariogenic biofilm models., Objective: Thus, this study aimed to evaluate the effects of EGCG on S. mutans biofilm virulence using a validated cariogenic model and clinically relevant treatment regimens, twice a day for 1.5 min., Methods: Effects of EGCG on bacterial viability, polyssacharide synthesis and biofilm acidogenesis were evaluated. The morphology and 3D structure of the biofilms were evaluated by scanning electron (SEM) and confocal laser scanning microscopy, respectively., Results: No significant change in S. mutans viability or culture medium pH were observed when comparing EGCG-treated and NaCl-treated biofilms. EGCG significantly reduced the accumulation of soluble and insoluble polysaccharides, resulting in the formation of a biofilm with interspaced exopolysaccharide-microcolony complexes unevenly distributed on enamel. The SEM images of the biofilm treated with EGCG depict multilayers of cells arranged in short chains of microorganisms adhered to an unstructured matrix, which is not continuous and does not enmesh or protect the microorganisms entirely. Importantly, confocal images demonstrated that treatment with EGCG affected the 3D structure and organization of S. mutans biofilm, which presented a biofilm matrix more confined to the location of the microcolonies., Conclusion: In conclusion, EGCG lowered the virulence of S. mutans matrix-rich biofilm by reducing the synthesis of biofilm matrix components, altering the biofilm matrix structure, organization, and distribution., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: The aim of this study was to describe the clinical features and endothelial involvement in a case of Mpox virus keratitis by in vivo confocal microscopy (IVCM)., Methods: This is a case report., Results: A 35-year-old man presented with redness, photophobia, pain, tearing, and a low visual acuity of 0.09 (decimal) in the left eye with a 6-week history of Mpox and corneal trauma. Previous testing of blood, interdigital skin lesions, and conjunctival and eyelid margin swabs confirmed the presence of Mpox by polymerase chain reaction. Biomicroscopy displayed superficial stromal infiltrates with a continuous but irregular epithelium. IVCM revealed the presence of pseudoguttata, loss of defined cell boundaries, infiltration of inflammatory cells in the endothelial layer, endothelial ridges, and precipitated pigmented granules, consistent with endotheliitis. After this episode, the patient had 4 reactivations, also treated with topical corticoids and oral tecovirimat 600 mg twice a day for 2 weeks. On the fourth reactivation, this treatment was extended to 4 weeks. On the last visit, the patient presented a visual acuity of 0.5 with disciform keratitis and reduced endotheliitis signs. The endothelial cell density remained normal during the follow-up (2763 ± 376 cell/mm 2 at baseline and 2795 ± 238 cell/mm 2 at the last visit). Polymegathism and pleomorphism showed altered values during the follow-up., Conclusions: Patients with an altered corneal epithelial barrier could suffer Mpox endotheliitis, like other DNA viruses, before disciform keratitis appears. IVCM is a useful tool for the early detection of endotheliitis and for describing its evolution, improving patient care., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Objective: Ex vivo skin has been used to study various skin conditions from atopic dermatitis to burn injury. The aim of this research is to identify a more effective barrier improvement strategy and to evaluate topical formulations in replenishing the skin. The skin can create new longer chain fatty acids and ceramides (CERs) from topically applied skin natural fatty acid to help renew the skin's barrier., Methods: An ex vivo skin model damaged by sequential tape stripping of the stratum corneum (SC) was used to investigate the repair of the SC. Confocal laser scanning microscopy was used to assess the SC layers recovered. Ultrastructural analysis was performed using transmission electron microscopy to visualize the lamellar bodies and intercellular lipid lamellae., Results: The data in this study provide the first direct ex vivo evidence comparing different marketed formulations containing three CERs with those containing fatty acids. Free fatty acid (FFA)-containing formulations, but not CER-containing formulations, directly applied to the damaged skin, showed an increased number of repaired SC layers and this was reflected at the ultrastructural level by an increased intercellular lipid lamellae length and an increased number of lamellar bodies., Conclusion: These findings demonstrate that FFA-containing formulations can repair damaged ex vivo skin and point to a repair mechanism in which topically applied palmitic and stearic acids, (which boost lipid levels and elongation) can increase the production and transport of lipids into a repaired SC and thus rebuild an effective skin barrier., (© 2024 Society of Cosmetic Scientists and Societe Francaise de Cosmetologie.)

In cereal products, the use of flour containing clusters of intact cells has been indicated as a potential strategy to decrease starch digestion. Rye possesses more uniform and thicker cell walls than wheat but its protective effect against starch digestion has not been elucidated. In this study, rye flours with three different particle sizes, large (LF) (∼1700 μm), medium (MF) (∼1200 μm), and small (SF) (∼350 μm), were used to produce model bread. The textural properties of these breads were analysed using Textural Profile Analysis (TPA). The starch digestibility of both the flour and the bread was measured using Englyst's method, while the presence of intact cell clusters was examined using Confocal Laser Scanning Microscopy (CLSM). Additionally, the disintegration of bread digesta during simulated digestion was assessed through image analysis. CLSM micrographs revealed that bread made with MF and LF retained clusters of intact cells after processing, whereas bread made with SF showed damaged cell walls. Starch digestibility in LF and MF was lower (p ≤ 0.05) than that in SF. Bread produced with MF and LF exhibited the least (p ≤ 0.05) cohesive and resilient texture, disintegrated more during digestion, and exhibited higher starch digestibility (p ≤ 0.05) than bread made with SF. These results highlight the central role of bread texture on in vitro starch digestibility., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Hydrolytically and enzymatically-stable multi-acrylamides have been proposed to increase the long-term durability of dental adhesive interfaces as alternatives to methacrylates. The aim of this study was to investigate the mechanical and biochemical properties of experimental adhesives containing multi-functional acrylamides concerning collagen reinforcement and metalloproteinases (MMP) activity. Multi-functional acrylamides, TMAAEA (Tris[(2-methylaminoacryl) ethylamine) and DEBAAP (N,N-Diethyl-1,3-bis(acrylamido) propane), along with the commercially available DMAM (N,N-dimethylacrylamide) (monofunctional acrylamide) and HEMA (2-Hydroxyethyl methacrylate) (monofunctional methacrylate - control) were tested for stability against enzymatic hydrolysis by cholesterol esterase/pseudocholinesterase (PC/PCE) solutions for up to 30 days. Collagen-derived substrate and gelatin zymography were performed to examine the effect of the compounds on the biological activity of human recombinant and dentin-extracted gelatinases MMP-2 and MMP-9. In situ zymography was carried out by fluorescent collagen degradation combined with confocal microscopy analysis. Hydroxyproline content was measured in collagen derived from dentin extracts though reaction with Ehrlich's reagent p-dimethylaminobenzaldehyde (DMAB), generating a stable chromophore measured at 550 nm. Storage shear modulus of demineralized dentin discs treated with the tested compounds was measured by oscillatory rheometry, in order to investigate potential collagen reinforcement. FT-IR was performed to determine qualitative differences in collagen based on observed changes in amide bands. The results were analyzed by ANOVA/Tukey's test (α = 0.05). Multi-acrylamides survived 30 days of incubation in cholinesterase/pseudo-cholinesterase (PC/PCE) solutions, while HEMA showed approximately 70 % overall degradation. Incubation with multi-acrylamides reduced collagen degradation as evidenced by the reduced hydroxyproline levels and by the 30 % increase inshear storage modulus. Biochemical and zymography assays showed no noticeable inhibition of recombinant and extracted MMPs enzymatic activity. The infra-red spectroscopy results for multi-functional acrylamides treated samples demonstrated shifts of the amide II bonds and marked increase in intensity of the bands 1200 cm -1 , which may indicate partial collagen denaturation and some degree of cross-linking of the compounds with collagen, respectively. The multi-acrylamides exhibited not only comparable mechanical properties but also demonstrated significantly enhanced biochemical stability when compared to the widely used methacrylate control. Clinical relevance: These findings highlight the potential of multi-acrylamides to increase the bonding stability to tissues and, ultimately, contribute to the longevity of dental restorations., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: The purpose of this study was to determine the association between corneal images provided by in vivo confocal microscopy (IVCM) with clinical parameters and conjunctival expression of HLA-DR antigen in patients with dry eye disease (DED)., Methods: Two hundred fourteen eyes of 214 patients with DED were analyzed, consisting of 2 groups of patients - 63 with autoimmune dry eye disease (AIDED) and 151 with non-autoimmune dry eye disease (NAIDED). Patients underwent a full clinical examination, including symptom screening, using the Ocular Surface Disease Index (OSDI) questionnaire, and objective analysis of DED signs by Schirmer's testing, tear break-up time (TBUT), Oxford's test, and IVCM corneal imaging. The IVCM scoring criteria were based on corneal sub-basal nerve density (ND), nerve morphology (NM), and inflammatory cell (IC) density. Quantification of conjunctival HLA-DR antigen was performed by flow cytometry., Results: The total IVCM score (T-IVCM) as well as the IVCM-IC subscore (sc) were positively correlated with HLA-DR levels with r = 0.3, P < 0.001 and r = 0.3, P < 0.01, respectively in the total population of patients with DED. The IVCM-NDsc was negatively correlated with TBUT in patients with AIDED (r = -0.2, P < 0.05) and with the Schirmer's test in patients with NAIDED (r = -0.24, P < 0.05). However, the IVCM-NMsc was positively correlated with the Oxford score only in patients with AIDED (r = 0.3, P < 0.05)., Conclusions: The proposed IVCM scoring system showed significant correlations with clinical parameters along with conjunctival HLA-DR quantification in patients with DED., Translational Relevance: The IVCM grading score represents an interesting point of commonality among clinical parameters, imaging, and molecular investigation of the ocular surface.

The study of the changes in rheological properties and components during the processing of Chinese traditional handmade hollow dried noodle (HHDN) is essential to explaining the excellent quality of HHDN. The dynamic oscillation frequency sweep, stress relaxation, and uniaxial extension characteristics of the dough after kneading, stretching, and resting were investigated at six sampling points during the processing of HHDN. The result showed that stretching led to an increase in G' and G 0 , and a significant decrease (P < 0.05) in extensibility from 131.02 mm to 57.99 mm. Confocal laser scanning microscopy (CLSM) was used to observe the microstructure of the gluten network, which was destroyed during stretching and restored during resting. Studies of changes in components showed that the stretching process resulted in a decrease in GMP content from 3.24 (g/100 g) to 3.18 (g/100 g), and the resting process resulted in β-sheets decreasing significantly (P < 0.05). The degree of starch pasting increased significantly (P < 0.05) after stretching. The results of the correlation analysis showed that components changes were highly correlated with the rheological properties during the processing of HHDN., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

RNA interference (RNAi) is a powerful and rapidly developing technology that enables precise silencing of genes of interest. However, the clinical development of RNAi is hampered by the limited cellular uptake and stability of the transferred molecules. Electroporation (EP) is an efficient and versatile technique for the transfer of both RNA and DNA. Although the mechanism of electrotransfer of small nucleic acids has been studied previously, too little is known about the potential effects of significantly larger pDNA on this process. Here we present a fundamental study of the mechanism of electrotransfer of oligonucleotides and siRNA that occur independently and simultaneously with pDNA by employing confocal fluorescence microscopy. In contrast to the conditional understanding of the mechanism, we have shown that the electrotransfer of oligonucleotides and siRNA is driven by both electrophoretic forces and diffusion after EP, followed by subsequent entry into the nucleus within 5 min after treatment. The study also revealed that the efficiency of siRNA electrotransfer decreases in response to an increase in pDNA concentration. Overall, the study provides new insights into the mechanism of electrotransfer of small nucleic acids which may have broader implications for the future application of RNAi-based strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Periodontal disease is the leading cause of tooth loss, and an association between periodontal disease and non-oral systemic diseases has been shown. Formation of biofilm by periodontal pathogens such as Fusobacterium nucleatum, Porphyromonas gingivalis, and Streptococcus mutans and their resistance to antimicrobial agents are at the root of persistent and chronic bacterial infections., Methods: The bactericidal effect of far-ultraviolet (F-UV) light irradiation at 222 nm on periodontal bacteria was assessed qualitatively and quantitatively. The effect of biofilm disruption by F-UV light on periodontal bacteria was examined by crystal violet staining, and the morphologic changes of the biofilm after F-UV irradiation were explored by confocal laser microscopy and scanning electron microscopy. We developed a thin fiber-type 222 nm F-UV irradiator and studied its safety and effect of reducing bacteria in rodent models., Results: F-UV light at 222 nm had a bactericidal effect on F. nucleatum, P. gingivalis, and S. mutans. Irradiation with F-UV light reduced the biofilm formed by the bacteria and sterilized them from within. Confocal laser microscopy showed a clear reduction in biofilm thickness, and scanning electron microscopy confirmed disintegration of the biofilm architecture. F-UV irradiation was less damaging to DNA and less cytotoxic than deep-ultraviolet light, and it reduced bacterial counts on the tooth surface., Conclusion: F-UV irradiation has the potential to destroy biofilm and act as a bactericide against pathogenic bacteria in the biofilm., Competing Interests: Declaration of competing interest J.N., T.F., S.F., and K.H. are the inventors of the device in the patent application (2022-073254) filed by Ushio Inc. and Yamaguchi University for biofilm dispersion by far-ultraviolet light described in this manuscript. The application is currently under examination. The other authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

Purpose: To investigate the morphological features of keratic precipitates (KPs) with in vivo confocal microscopy (IVCM) in vitreoretinal lymphoma (VRL)., Methods: Patients with biopsy-proven VRL were reviewed, and 12 patients (16 eyes) with KPs examined by IVCM were included., Results: Five distinct KP morphologies on IVCM were observed: dendritic, nibbling, linear, globular, and stippled. Dendritic KPs were discovered in 9 eyes (56.3%), with a hyperreflective core and multiple thin pseudopodia. Nibbling KPs were found in 8 eyes (50.0%), featuring irregularly hyperreflective borders and a mottled reflective interior. Linear, stippled, and globular KPs were self-explanatory and affected nearly all eyes. Globular KPs seem to be formed by hyperreflective cells with large nuclei and prominent nucleoli, similar to atypical lymphocytes in vitreous cytology., Conclusions: IVCM could provide more insight into the morphological traits of KPs than clinical observation, highlighting its potential for identifying VRL involving the anterior segment.

Aims/introduction: Deficiency of neurotropic factors is implicated in diabetic neuropathy (DN). Netrin-1 is a neurotropic factor, but its association with DN has not been explored. We have assessed the association between serum netrin-1 levels and early diabetic neuropathy assessed by quantifying corneal nerve fiber loss using corneal confocal microscopy., Materials and Methods: A total of 72 participants with type 2 diabetes, without and with corneal nerve fiber loss (DN- n = 42, DN+ n = 30), and 45 healthy controls were studied. Serum netrin-1 levels were measured by enzyme-linked immunosorbent assay, and corneal nerve morphology was assessed using corneal confocal microscopy., Results: Corneal nerve fiber density, branch density, fiber length and serum netrin-1 levels were significantly lower in the DN- and DN+ groups compared with controls (P < 0.001). Netrin-1 levels correlated with corneal nerve fiber length in the DN+ group (r = 0.51; P < 0.01). A receiver operating characteristic curve analysis showed that a netrin-1 cut-off value of 599.6 (pg/mL) had an area under the curve of 0.85, with a sensitivity of 76% and specificity of 74% (P < 0.001; 95% confidence interval 0.76-0.94) for differentiating patients with and without corneal nerve loss., Conclusions: Serum netrin-1 levels show a progressive decline with increasing severity of small nerve fiber damage in patients with diabetes. Netrin-1 could act as a biomarker for small nerve fiber damage in DN., (© 2024 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

The high prevalence of dental caries and periodontal disease place a significant burden on society, both socially and economically. Recent advances in genomic technologies have linked both diseases to shifts in the oral microbiota - a community of >700 bacterial species that live within the mouth. The development of oral microbiome transplantation draws on the success of fecal microbiome transplantation for the treatment of gut pathologies associated with disease. Many current in vitro oral biofilm models have been developed but do not fully capture the complexity of the oral microbiome which is required for successful OMT. To address this, we developed an in vitro biofilm system that maintained an oral microbiome with 252 species on average over 14 days. Six human plaque samples were grown in 3D printed flow cells on hydroxyapatite discs using artificial saliva medium (ASM). Biofilm composition and growth were monitored by high throughput sequencing and confocal microscopy/SEM, respectively. While a significant drop in bacterial diversity occurred, up to 291 species were maintained in some flow cells over 14 days with 70% viability grown with ASM. This novel in vitro biofilm model represents a marked improvement on existing oral biofilm systems and provides new opportunities to develop oral microbiome transplant therapies., Competing Interests: Declaration of competing interest All authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: To assess the long-term (1-year) effect of myopic femtosecond laser-assisted in situ keratomileusis (FSLASIK) on clinical characteristics and tear film biomarkers., Methods: Eighty eyes from 80 patients who underwent FSLASIK were evaluated. Ocular surface symptoms and signs were evaluated using specific questionnaires and tests. The corneal nerves and dendritic cells were examined using in vivo confocal microscopy. Corneal sensitivity was evaluated using a Cochet-Bonnet esthesiometer. Tear inflammatory cytokines and neuropeptides were evaluated using Luminex immunoassay. These examinations were performed preoperatively and at 1, 3, 6, and 12 months postoperatively., Results: Seventy-three participants completed all follow-up visits. Following FS-LASIK, ocular symptoms and signs (except Schirmer I test) worsened at 1 month but corneal and conjunctival stainings improved by 3 months. The numbers of dendritic cells and activated dendritic cells increased at the 3-month postoperative visit and recovered to preoperative levels by the 6-month visit. Ocular symptoms and corneal sensitivity recovered to preoperative levels at the 12-month visit. Tear break-up time and corneal nerve morphology were not recovered to preoperative status at the 12-month visit. Interleukin (IL)-1β, IL-17A, tumor necrosis factor-α, and substance P tear levels significantly increased at all postoperative visits compared to preoperative levels. Corneal staining scores positively correlated with tear IL-1β and IL-17A levels, whereas corneal nerve morphology positively correlated with corneal sensitivity and negatively correlated with substance P levels., Conclusions: Although most clinical variables improved at 12 months postoperatively, some tear inflammatory cytokines and substance P remain altered beyond 12 months, indicating that ocular homeostasis is not completely recovered. [ J Refract Surg . 2024;40(8):e508-e519.] .