Using long-read CAGE sequencing to profile cryptic-promoter-derived transcripts and their contribution to the immunopeptidome.

Recent studies have shown that the noncoding genome can produce unannotated proteins as antigens that induce immune response. One major source of this activity is the aberrant epigenetic reactivation of transposable elements (TEs). In tumors, TEs often provide cryptic or alternate promoters, which can generate transcripts that encode tumor-specific unannotated proteins. Thus, TE-derived transcripts (TE transcripts) have the potential to produce tumor-specific, but recurrent, antigens shared among many tumors. Identification of TE-derived tumor antigens holds the promise to improve cancer immunotherapy approaches; however, current genomics and computational tools are not optimized for their detection. Here we combined CAGE technology with full-length long-read transcriptome sequencing (long-read CAGE, or LRCAGE) and developed a suite of computational tools to significantly improve immunopeptidome detection by incorporating TE and other tumor transcripts into the proteome database. By applying our methods to human lung cancer cell line H1299 data, we show that long-read technology significantly improves mapping of promoters with low mappability scores and that LRCAGE guarantees accurate construction of uncharacterized 5' transcript structure. Augmenting a reference proteome database with newly characterized transcripts enabled us to detect noncanonical antigens from HLA-pulldown LC-MS/MS data. Lastly, we show that epigenetic treatment increased the number of noncanonical antigens, particularly those encoded by TE transcripts, which might expand the pool of targetable antigens for cancers with low mutational burden.

Determinants of Meal-Induced Changes in Circulating FFA Epoxides, Diols, and Diol-to-Epoxide Ratios as Indices of Soluble Epoxide Hydrolase Activity.

Soluble epoxide hydrolase (sEH) is an important enzyme for metabolic and cardiovascular health. sEH converts FFA epoxides (EpFAs), many of which are regulators of various cellular processes, to biologically less active diols. In human studies, diol (sEH product) to EpFA (sEH substrate) ratios in plasma or serum have been used as indices of sEH activity. We previously showed these ratios profoundly decreased in rats during acute feeding, possibly reflecting decreases in tissue sEH activities. The present study was designed to test which tissue(s) these measurements in the blood represent and if factors other than sEH activity, such as renal excretion or dietary intake of EpFAs and diols, significantly alter plasma EpFAs, diols, and/or their ratios. The results show that postprandial changes in EpFAs and diols and their ratios in plasma were very similar to those observed in the liver but not in other tissues, suggesting that the liver is largely responsible for these changes in plasma levels. EpFAs and diols were excreted into the urine, but their levels were not significantly altered by feeding, suggesting that renal excretion of EpFAs and diols may not play a major role in postprandial changes in circulating EpFAs, diols, or their ratios. Diet intake had significant impacts on circulating EpFA and diol levels but not on diol-to-EpFA (D-to-E) ratios, suggesting that these ratios, reflecting sEH activities, may not be significantly affected by the availability of sEH substrates (i.e., EpFAs). In conclusion, changes in FFA D-to-E ratios in plasma may reflect those in the liver, which may in turn represent sEH activities in the liver, and they may not be significantly affected by renal excretion or the dietary intake of EpFAs and diols.

An evolutionary epigenetic clock in plants.

Molecular clocks are the basis for dating the divergence between lineages over macroevolutionary timescales (~105 to 108 years). However, classical DNA-based clocks tick too slowly to inform us about the recent past. Here, we demonstrate that stochastic DNA methylation changes at a subset of cytosines in plant genomes display a clocklike behavior. This "epimutation clock" is orders of magnitude faster than DNA-based clocks and enables phylogenetic explorations on a scale of years to centuries. We show experimentally that epimutation clocks recapitulate known topologies and branching times of intraspecies phylogenetic trees in the self-fertilizing plant Arabidopsis thaliana and the clonal seagrass Zostera marina, which represent two major modes of plant reproduction. This discovery will open new possibilities for high-resolution temporal studies of plant biodiversity.

Effects of Individual Circulating FFAs on Plasma and Hepatic FFA Epoxides, Diols, and Epoxide-Diol Ratios as Indices of Soluble Epoxide Hydrolase Activity.

Oxylipins, oxidation products of unsaturated free fatty acids (FFAs), are involved in various cellular signaling systems. Among these oxylipins, FFA epoxides are associated with beneficial effects in metabolic and cardiovascular health. FFA epoxides are metabolized to diols, which are usually biologically less active, by soluble epoxide hydrolase (sEH). Plasma epoxide-diol ratios have been used as indirect measures of sEH activity. This study was designed to examine the effects of acute elevation of individual plasma FFAs on a variety of oxylipins, particularly epoxides, diols, and their ratios. We tested if FFA epoxide-diol ratios are altered by circulating FFA levels (i.e., substrate availability) independent of sEH activity. Wistar rats received a constant intravenous infusion of olive (70% oleic acid (OA)), safflower seed (72% linoleic acid (LA)), and fish oils (rich in ω-3 FFAs) as emulsions to selectively raise OA, LA, and ω-3 FFAs (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), respectively. As expected, olive, safflower seed, and fish oil infusions selectively raised plasma OA (57%), LA (87%), EPA (70%), and DHA (54%), respectively (p < 0.05 for all). Raising plasma FFAs exerted substrate effects to increase hepatic and plasma epoxide and diol levels. These increases in epoxides and diols occurred to similar extents, resulting in no significant changes in epoxide-diol ratios. These data suggest that epoxide-diol ratios, often used as indices of sEH activity, are not affected by substrate availability or altered plasma FFA levels and that epoxide-diol ratios may be used to compare sEH activity between conditions of different circulating FFA levels.

Water in the terrestrial planet-forming zone of the PDS 70 disk.

Terrestrial and sub-Neptune planets are expected to form in the inner (less than 10 AU) regions of protoplanetary disks1. Water plays a key role in their formation2-4, although it is yet unclear whether water molecules are formed in situ or transported from the outer disk5,6. So far Spitzer Space Telescope observations have only provided water luminosity upper limits for dust-depleted inner disks7, similar to PDS 70, the first system with direct confirmation of protoplanet presence8,9. Here we report JWST observations of PDS 70, a benchmark target to search for water in a disk hosting a large (approximately 54 AU) planet-carved gap separating an inner and outer disk10,11. Our findings show water in the inner disk of PDS 70. This implies that potential terrestrial planets forming therein have access to a water reservoir. The column densities of water vapour suggest in-situ formation via a reaction sequence involving O, H2 and/or OH, and survival through water self-shielding5. This is also supported by the presence of CO2 emission, another molecule sensitive to ultraviolet photodissociation. Dust shielding, and replenishment of both gas and small dust from the outer disk, may also play a role in sustaining the water reservoir12. Our observations also reveal a strong variability of the mid-infrared spectral energy distribution, pointing to a change of inner disk geometry.

Pan-cancer analysis identifies tumor-specific antigens derived from transposable elements.

Cryptic promoters within transposable elements (TEs) can be transcriptionally reactivated in tumors to create new TE-chimeric transcripts, which can produce immunogenic antigens. We performed a comprehensive screen for these TE exaptation events in 33 TCGA tumor types, 30 GTEx adult tissues and 675 cancer cell lines, and identified 1,068 TE-exapted candidates with the potential to generate shared tumor-specific TE-chimeric antigens (TS-TEAs). Whole-lysate and HLA-pulldown mass spectrometry data confirmed that TS-TEAs are presented on the surface of cancer cells. In addition, we highlight tumor-specific membrane proteins transcribed from TE promoters that constitute aberrant epitopes on the extracellular surface of cancer cells. Altogether, we showcase the high pan-cancer prevalence of TS-TEAs and atypical membrane proteins that could potentially be therapeutically exploited and targeted.

A Phase II Trial of Guadecitabine plus Atezolizumab in Metastatic Urothelial Carcinoma Progressing after Initial Immune Checkpoint Inhibitor Therapy.

PURPOSE: On the basis of preclinical evidence of epigenetic contribution to sensitivity and resistance to immune checkpoint inhibitors (ICI), we hypothesized that guadecitabine (hypomethylating agent) and atezolizumab [anti-programmed cell death ligand 1 (PD-L1)] together would potentiate a clinical response in patients with metastatic urothelial carcinoma (UC) unresponsive to initial immune checkpoint blockade therapy. PATIENTS AND METHODS: We designed a single arm phase II study (NCT03179943) with a safety run-in to identify the recommended phase II dose of the combination therapy of guadecitabine and atezolizumab. Patients with recurrent/advanced UC who had previously progressed on ICI therapy with programmed cell death protein 1 or PD-L1 targeting agents were eligible. Preplanned correlative analysis was performed to characterize peripheral immune dynamics and global DNA methylation, transcriptome, and immune infiltration dynamics of patient tumors. RESULTS: Safety run-in enrolled 6 patients and phase II enrolled 15 patients before the trial was closed for futility. No dose-limiting toxicity was observed. Four patients, with best response of stable disease (SD), exhibited extended tumor control (8-11 months) and survival (>14 months). Correlative analysis revealed lack of DNA demethylation in tumors after 2 cycles of treatment. Increased peripheral immune activation and immune infiltration in tumors after treatment correlated with progression-free survival and SD. Furthermore, high IL6 and IL8 levels in the patients' plasma was associated with short survival. CONCLUSIONS: No RECIST responses were observed after combination therapy in this trial. Although we could not detect the anticipated tumor-intrinsic effects of guadecitabine, the addition of hypomethylating agent to ICI therapy induced immune activation in a few patients, which associated with longer patient survival.

Optimization of spray drying process of Japanese apricot (Prunus mume Sieb. et Zucc.) juice powder using nondigestible maltodextrin by response surface methodology (RSM).

The optimal spray-drying conditions for manufacturing Japanese apricot (Prunus mume Sieb. et Zucc.) juice powder (JAJP) using response surface methodology (RSM) were investigated. The optimization was performed using two independent factors, which are inlet air temperature (130-180 °C) and different concentrations of nondigestible maltodextrin (NMD) as a carrier agent (10-30%). Responses such as drying yield, moisture content, water solubility index (WSI), bulk density, color, pH, and antioxidant activities of JAJP were investigated. Moisture content, vitamin C content, color, antioxidant activity, pH and bulk density were greatly influenced by inlet air temperature, but dry yield and WSI were only significantly affected by NMD concentration. The optimum spray drying conditions were determined as 14.7% NMD concentration and 154.5 °C inlet air temperature, respectively. At these optimum conditions, a drying yield of 55.73%, 4.84% moisture content, 90.98% WSI, 0.59 g/mL of bulk density, and 169.87 mg/g vitamin C content in JAJP were measured. Therefore, JAJP with the desirable physicochemical properties could be produced.

An evolutionary epigenetic clock in plants.

Molecular clocks are the basis for dating the divergence between lineages over macro-evolutionary timescales (~10 5 -10 8 years). However, classical DNA-based clocks tick too slowly to inform us about the recent past. Here, we demonstrate that stochastic DNA methylation changes at a subset of cytosines in plant genomes possess a clock-like behavior. This 'epimutation-clock' is orders of magnitude faster than DNA-based clocks and enables phylogenetic explorations on a scale of years to centuries. We show experimentally that epimutation-clocks recapitulate known topologies and branching times of intra-species phylogenetic trees in the selfing plant A. thaliana and the clonal seagrass Z. marina , which represent two major modes of plant reproduction. This discovery will open new possibilities for high-resolution temporal studies of plant biodiversity.

Enhanced charge density wave with mobile superconducting vortices in La1.885Sr0.115CuO4.

Superconductivity in the cuprates is found to be intertwined with charge and spin density waves. Determining the interactions between the different types of order is crucial for understanding these important materials. Here, we elucidate the role of the charge density wave (CDW) in the prototypical cuprate La1.885Sr0.115CuO4, by studying the effects of large magnetic fields (H) up to 24 Tesla. At low temperatures (T), the observed CDW peaks reveal two distinct regions in the material: a majority phase with short-range CDW coexisting with superconductivity, and a minority phase with longer-range CDW coexisting with static spin density wave (SDW). With increasing magnetic field, the CDW first grows smoothly in a manner similar to the SDW. However, at high fields we discover a sudden increase in the CDW amplitude upon entering the vortex-liquid state. Our results signify strong coupling of the CDW to mobile superconducting vortices and link enhanced CDW amplitude with local superconducting pairing across the H - T phase diagram.

Efficiency analysis by training sequence of high-fidelity simulation-based neonatal resuscitation program (NRP).

OBJECTIVE: This study was conducted to find a more effective education method in a neonatal resuscitation program (NRP) using a high-fidelity simulation that has recently shown positive results in terms of its effectiveness. STUDY DESIGN: A single-blind prospective cohort study was performed. The high-fidelity simulation model was used in a formal NRP training course for trainees caring for neonatal patients. The trainees were divided into a group that conducted the scenario after the lecture (Group A) and a group that attended the lecture after the scenario (Group B) and they both took the test before, during, and after the training. RESULTS: The increase in score after theory training was statistically significant in both groups, but the final score did not differ between the two groups. However, when compared by career, in Group A, trainees under 24 months tended to be more effective, and in Group B, trainees over 24 months tended to be more effective. CONCLUSION: The difference in short-term memory of trainees according to the order of education identified by the test score was not prominent, but it was found that the degree of difference in test scores for the order of education tended to be different according to the career. It is thought that the effectiveness of the training might be increased by changing the education order according to the degree of experience of each trainee. More effective educational methods should be introduced by continuously developing lectures for repeated education of various trainees in the future.

RNA mis-splicing drives viral mimicry response after DNMTi therapy in SETD2-mutant kidney cancer.

Tumors with mutations in chromatin regulators present attractive targets for DNA hypomethylating agent 5-aza-2'-deoxycytidine (DAC) therapy, which further disrupts cancer cells' epigenomic fidelity and reactivates transposable element (TE) expression to drive viral mimicry responses. SETD2 encodes a histone methyltransferase (H3K36me3) and is prevalently mutated in advanced kidney cancers. Here, we show that SETD2-mutant kidney cancer cells are especially sensitive in vitro and in vivo to DAC treatment. We find that the viral mimicry response are direct consequences of mis-splicing events, such as exon inclusions or extensions, triggered by DAC treatment in an SETD2-loss context. Comprehensive epigenomic analysis reveals H3K9me3 deposition, rather than DNA methylation dynamics, across intronic TEs might contribute to elevated mis-splicing rates. Through epigenomic and transcriptomic analyses, we show that SETD2-deficient kidney cancers are prone to mis-splicing, which can be therapeutically exacerbated with DAC treatment to increase viral mimicry activation and provide synergy with combinatorial immunotherapy approaches.

Prevalence of preoperative anxiety and its contributing factors in adult patients undergoing elective orthopaedic surgery at an academic hospital in South Africa: a pilot study

Preoperative anxiety potentially results in perioperative anaesthetic complications. This study aimed to determine the prevalence of preoperative anxiety in adult patients scheduled to undergo elective orthopaedic surgery at an academic hospital in South Africa and identify contributory factors. Methods: This cross-sectional study included all patients scheduled for elective orthopaedic surgery the following day from 25 July to 3 November 2021. An adapted version of the Amsterdam Preoperative Anxiety and Information Scale (APAIS) was used. Four questions determined the patients' anxiety and two questions their desire for more information about their forthcoming anaesthesia and surgery. A 5-point Likert scale was used to capture the patients' responses to the APAIS questions. Data on other possible contributory factors to preoperative anxiety were collected using a questionnaire. Results: Of the 88 patients, 82 (93.2%) were included in the study. A total of 40 (48.8%) patients had an APAIS score of ≥ 11, indicating anxiety. A high need-for-information score (≥ 8) was found in 31 (37.8%) patients. A significant association between patients with a higher anxiety score and a higher need-for-information score (p = 0.0063) was identified. Other non-significant factors associated with a higher anxiety score included a lower level of education and no postoperative home support. Conclusion: Patients with a high need for information tended to be more anxious on the day before surgery. The APAIS could be introduced in preoperative ward admissions to identify these patients and provide an appropriate level of counselling about their planned procedure. Counselling might reduce their preoperative anxiety, but further research needs to confirm this assertion. Larger studies are recommended to determine the influence of other factors contributing to preoperative anxiety

Stable potassium isotopes (41K/39K) track transcellular and paracellular potassium transport in biological systems.

As the most abundant cation in archaeal, bacterial, and eukaryotic cells, potassium (K+) is an essential element for life. While much is known about the machinery of transcellular and paracellular K transport-channels, pumps, co-transporters, and tight-junction proteins-many quantitative aspects of K homeostasis in biological systems remain poorly constrained. Here we present measurements of the stable isotope ratios of potassium (41K/39K) in three biological systems (algae, fish, and mammals). When considered in the context of our current understanding of plausible mechanisms of K isotope fractionation and K+ transport in these biological systems, our results provide evidence that the fractionation of K isotopes depends on transport pathway and transmembrane transport machinery. Specifically, we find that passive transport of K+ down its electrochemical potential through channels and pores in tight-junctions at favors 39K, a result which we attribute to a kinetic isotope effect associated with dehydration and/or size selectivity at the channel/pore entrance. In contrast, we find that transport of K+ against its electrochemical gradient via pumps and co-transporters is associated with less/no isotopic fractionation, a result that we attribute to small equilibrium isotope effects that are expressed in pumps/co-transporters due to their slower turnover rate and the relatively long residence time of K+ in the ion pocket. These results indicate that stable K isotopes may be able to provide quantitative constraints on transporter-specific K+ fluxes (e.g., the fraction of K efflux from a tissue by channels vs. co-transporters) and how these fluxes change in different physiological states. In addition, precise determination of K isotope effects associated with K+ transport via channels, pumps, and co-transporters may provide unique constraints on the mechanisms of K transport that could be tested with steered molecular dynamic simulations.

Temporal control of PDGFRα regulates the fibroblast-to-myofibroblast transition in wound healing.

Fibroblasts differentiate into myofibroblasts by acquiring new contractile function. This is important for tissue repair, but it also contributes to organ fibrosis. Platelet-derived growth factor (PDGF) promotes tissue repair and fibrosis, but the relationship between PDGF and myofibroblasts is unclear. Using mice with lineage tracing linked to PDGF receptor α (PDGFRα) gene mutations, we examine cell fates during skin wound healing. Elevated PDGFRα signaling increases proliferation but unexpectedly delays the fibroblast-to-myofibroblast transition, suggesting that PDGFRα must be downregulated for myofibroblast differentiation. In contrast, deletion of PDGFRα decreases proliferation and myofibroblast differentiation by reducing serum response factor (SRF) nuclear localization. Consequences of SRF deletion resemble PDGFRα deletion, but deletion of two SRF coactivators, MRTFA and MRTFB, specifically eliminates myofibroblasts. Our findings suggest a scenario where PDGFRα signaling initially supports proliferation of fibroblast progenitors to expand their number during early wound healing but, later, PDGFRα downregulation facilitates fibroblast differentiation into myofibroblasts.

CRISPR activation screen identifies BCL-2 proteins and B3GNT2 as drivers of cancer resistance to T cell-mediated cytotoxicity.

The cellular processes that govern tumor resistance to immunotherapy remain poorly understood. To gain insight into these processes, here we perform a genome-scale CRISPR activation screen for genes that enable human melanoma cells to evade cytotoxic T cell killing. Overexpression of four top candidate genes (CD274 (PD-L1), MCL1, JUNB, and B3GNT2) conferred resistance in diverse cancer cell types and mouse xenografts. By investigating the resistance mechanisms, we find that MCL1 and JUNB modulate the mitochondrial apoptosis pathway. JUNB encodes a transcription factor that downregulates FasL and TRAIL receptors, upregulates the MCL1 relative BCL2A1, and activates the NF-κB pathway. B3GNT2 encodes a poly-N-acetyllactosamine synthase that targets >10 ligands and receptors to disrupt interactions between tumor and T cells and reduce T cell activation. Inhibition of candidate genes sensitized tumor models to T cell cytotoxicity. Our results demonstrate that systematic gain-of-function screening can elucidate resistance pathways and identify potential targets for cancer immunotherapy.

Estimating in vivo potassium distribution and fluxes with stable potassium isotopes.

Extracellular potassium (K+) homeostasis is achieved by a concerted effort of multiple organs and tissues. A limitation in studies of K+ homeostasis is inadequate techniques to quantify K+ fluxes into and out of organs and tissues in vivo. The goal of the present study was to test the feasibility of a novel approach to estimate K+ distribution and fluxes in vivo using stable K+ isotopes. 41K was infused as KCl into rats consuming control or K+-deficient chow (n = 4 each), 41K-to-39K ratios in plasma and red blood cells (RBCs) were measured by inductively coupled plasma mass spectrometry, and results were subjected to compartmental modeling. The plasma 41K/39K increased during 41K infusion and decreased upon infusion cessation, without altering plasma total K+ concentration ([K+], i.e., 41K + 39K). The time course of changes was analyzed with a two-compartmental model of K+ distribution and elimination. Model parameters, representing transport into and out of the intracellular pool and renal excretion, were identified in each rat, accurately predicting decreased renal K+ excretion in rats fed K+-deficient vs. control diet (P < 0.05). To estimate rate constants of K+ transport into and out of RBCs, 41K/39K were subjected to a simple model, indicating no effects of the K+-deficient diet. The findings support the feasibility of the novel stable isotope approach to quantify K+ fluxes in vivo and sets a foundation for experimental protocols using more complex models to identify heterogeneous intracellular K+ pools and to answer questions pertaining to K+ homeostatic mechanisms in vivo.

Oral Candida Predicts Streptococcus mutans Emergence in Underserved US Infants.

Despite the cariogenic role of Candida suggested from recent studies, oral Candida acquisition in children at high risk for early childhood caries (ECC) and its association with cariogenic bacteria Streptococcus mutans remain unclear. Although ECC disproportionately afflicts socioeconomically disadvantaged and racial-minority children, microbiological studies focusing on the underserved group are scarce. Our prospective cohort study examined the oral colonization of Candida and S. mutans among 101 infants exclusively from a low-income and racial-minority background in the first year of life. The Cox hazard proportional model was fitted to assess factors associated with the time to event of the emergence of oral Candida and S. mutans. Oral Candida colonization started as early as 1 wk among 13% of infants, increased to 40% by 2 mo, escalated to 48% by 6 mo, and remained the same level until 12 mo. S. mutans in saliva was detected among 20% infants by 12 mo. The emergence of S. mutans by year 1 was 3.5 times higher (hazard ratio [HR], 3.5; confidence interval [CI], 1.1-11.3) in infants who had early colonization of oral Candida compared to those who were free of oral Candida (P = 0.04) and 3 times higher (HR, 3.0; CI, 1.3-6.9) among infants whose mother had more than 3 decayed teeth (P = 0.01), even after adjusting demographics, feeding, mother's education, and employment status. Infants' salivary S. mutans abundance was positively correlated with infants' Candida albicans (P < 0.01) and Candida krusei levels (P < 0.05). Infants' oral colonization of C. albicans was positively associated with mother's oral C. albicans carriage and education (P < 0.01) but negatively associated with mother's employment status (P = 0.01). Future studies are warranted to examine whether oral Candida modulates the oral bacterial community as a whole to become cariogenic during the onset and progression of ECC, which could lead to developing novel ECC predictive and preventive strategies from a fungal perspective.

Skeletal stem cell fate defects caused by Pdgfrb activating mutation.

Autosomal dominant PDGFRß gain-of-function mutations in mice and humans cause a spectrum of wasting and overgrowth disorders afflicting the skeleton and other connective tissues, but the cellular origin of these disorders remains unknown. We demonstrate that skeletal stem cells (SSCs) isolated from mice with a gain-of-function D849V point mutation in PDGFRß exhibit colony formation defects that parallel the wasting or overgrowth phenotypes of the mice. Single-cell RNA transcriptomics with SSC-derived polyclonal colonies demonstrates alterations in osteogenic and chondrogenic precursors caused by PDGFRßD849V. Mutant cells undergo poor osteogenesis in vitro with increased expression of Sox9 and other chondrogenic markers. Mice with PDGFRßD849V exhibit osteopenia. Increased STAT5 phosphorylation and overexpression of Igf1 and Socs2 in PDGFRßD849V cells suggests that overgrowth in mice involves PDGFRßD849V activating the STAT5-IGF1 axis locally in the skeleton. Our study establishes that PDGFRßD849V causes osteopenic skeletal phenotypes that are associated with intrinsic changes in SSCs, promoting chondrogenesis over osteogenesis.

Optimal application of compressive palatal stents following mesiodens removal in pediatric patients: A Randomized Controlled Trial.

BACKGROUND: There is no scientific evidence supporting the choice of a palatal stent in patients who underwent removal of an impacted supernumerary tooth. We aimed to investigate the effects of palatal stents in patients who underwent supernumerary tooth removal through a palatal approach and to suggest the optimal stent thickness and material. MATERIAL AND METHODS: We recruited 144 patients who underwent extraction of a supernumerary tooth between the maxillary anterior teeth. Subjects were assigned to a control group (CG) or one of four compressive palatal stent groups (CPSGs) classified by the thickness and material of the thermoplastic acrylic stent used. Palatal gingival swelling and objective indices (healing, oral hygiene, gingival, and plaque) were evaluated before surgery and on postoperative days (PODs) 3, 7, and 14; pain/discomfort and the Child Oral Health Impact Profile (COHIP) were assessed as subjective indices of the effects of the stent. RESULTS: The CPSGs showed faster healing than did the CG on PODs 7 (P<0.001) and 14 (P=0.043); swelling was measured by 1.64±0.88 mm and 4.52±0.39 mm, respectively. Although swelling was least in the 4-mm hard group (0.92±0.33 mm), the difference compared with that in the 2-mm hard group (1.01±0.18 mm) was not significant (P=0.077). The CPSGs showed better COHIP (P<0.001-0.036) and pain scores (P<0.001) than did the CG on PODs 1-3. CONCLUSIONS: Compressive palatal stents reduce discomfort by decreasing pain and alleviating swelling. Although a stent is effective regardless of its thickness and material, 2-mm hard stents maximized such positive effects with minimal discomfort.