Biomolecules

Background: Higher education has been transformed by the rapid integration of generative artificial intelligence (GenAI) tools into academia. The objective of the present study was to examine how and for what purposes senior undergraduate dental students use GenAI tools in academic assignments. Methods: This cross-sectional study uses data from three written assignments submitted by two consecutive cohorts of graduating fourth-year dental students at the Faculty of Dentistry at the University of British Columbia, for a total of 120 students. The assignments focused on different subjects where students had to offer their views, including community water fluoridation. When using GenAI, students were asked to disclose whether and how such tools were used, and for what purpose. Descriptive statistics (e.g., means, frequencies, and proportions) were conducted via IBM SPSS Statistics (Version 27.0). Results: From the two cohort of students, 102 (85%) disclosed the use of GenAI tools in at least one assignment; of these, 69 (67.6%) reported using these tools in all three assignments. ChatGPT was by far the most frequently used GenAI tool, reported by 89 students (87.2%). Nine students (8.8%) did not specify which tool they had used. The majority of the students (91.2%, n = 93) reported using GenAI for proofreading or grammatical editing. About 9.8% of the students (n = 10) reported more substantive uses, such as relying on GenAI to generate in part or in full the assignment, and/or assessing the credibility of references. Conclusions: In our study, the use of GenAI tools was highly prevalent among senior undergraduate dental students for editorial purposes. A smaller but notable proportion of students engaged in more substantive uses that may carry academic and ethical risks. There is a need for structured AI literacy training and clear, dentistry-specific guidelines to promote responsible and transparent use while safeguarding critical thinking, academic integrity, and professional judgment in dental education.

Statins, widely used cholesterol-lowering drugs, inhibit the mevalonate pathway and reduce coenzyme Q (CoQ) biosynthesis, potentially impairing mitochondrial function. Because astrocytes are essential for maintaining brain redox homeostasis, statin-induced mitochondrial dysfunction in these cells may contribute to CNS pathology. We examined the effects of a six-day statin exposure on mitochondrial bioenergetics in rat astrocytes, focusing on mitochondrial CoQ (mtCoQ) deficiency. Treatment with 200 nM atorvastatin or simvastatin decreased the total mtCoQ pool (mtCoQ9 + mtCoQ10) by 30-35% and decreased the antioxidant pool mtCoQH2 by 40%, whereas the levels of mitochondrial antioxidant proteins, including superoxide dismutase 2 and uncoupling proteins, remained unchanged. Mitochondria of statin-treated astrocytes showed decreased respiratory activity, membrane potential, and ATP synthesis, and increased mtCoQ reduction leading to increased H2O2 production during the oxidation of complex I (CI) and CII substrates. Statin treatment also altered the organization of the respiratory chain, leading to a downregulation of the CI+CIII2+CIV and CIII2+CIV supercomplexes and decreased protein levels and activity of all respiratory chain complexes. Furthermore, a decrease in cytochrome a + a3 content was accompanied by a reduction in the maximum activity of CIV. CoQ10 supplementation elevated mtCoQ levels, restored respiratory function, and decreased H2O2 production in the mitochondria of statin-treated astrocytes. Prolonged statin exposure alters mtCoQ redox homeostasis and impairs mitochondrial bioenergetic function in astrocytes. CoQ10 supplementation attenuates these changes, supporting its potential role in protecting astrocyte mitochondria from statin-induced dysfunction.

BACKGROUND: Erythrophagocytosis constitutes a major pathogenic mechanism of metabolic dysfunction associated fatty liver disease (MAFLD). Our previous research established a quantitative thin-layer chromatography (TLC) technique for sphingomyelin, revealing reduced levels in the red blood cells (erythrocytes) of patients with metabolic dysfunction associated fatty liver disease (MAFLD). This reduction was accompanied by erythrocyte sphingosine accumulation, a driver of pro-inflammatory erythrophagocytosis, though sphingosine 1-phosphate release remained stable. To better understand erythrocyte sphingosine metabolism, we adapted our quantitative TLC method to analyze sphingosine within the erythrocyte-conditioned media (ECM) of MAFLD patients. Methodology Separation was performed on 10X10cm Silica gel 60 F254 plates using a mobile phase of chloroform, methanol, acetic acid, and water (60:50:1:4 v/v/v/v). The dynamic range, linearity, and range of linearity were assessed by analysing sphingosine levels from 0.1 to 10microg/spot. We validated the system precision and sensitivity by performing triplicate analyses of sphingosine standards (1.25, 2.5, and microg). The limits of detection and quantification were derived from the calibration curve slope and standard deviation (3.3 XSD/slope for LOD; 10 XSD/slope for LOQ). Accuracy was assessed via recovery tests at 100%, 200%, and 300% of a 2.5microg load. We confirmed specificity by evaluating the retention factors against other lipid species. This protocol was applied to Folch-extracted lipids from the ECM (5 X 107 cells/ml) of four MAFLD patients and four healthy controls, spiked with 5microg of sphingosine. Findings The calibration model, based on combined Green and Blue color intensities, followed the linear equation y = -11.171x + 353.25(R2 = 0.94). Interday precision values were 0.21%, 1.65%, and 0.44%, while recovery rates (accuracy) ranged from 94.5% to 98.7%. The measured LOD and LOQ were 0.75microg and 1.21microg, respectively. The sensitivity was calculated at 90ng. Statistical analysis showed no significant variance in sphingosine concentrations in erythrocyte-conditioned media between the MAFLD group and the control group. Summary The described thin layer chromatography is accurate, precise, sensitive, with good limits of detection and quantification, and most importantly is low-cost and time-efficient. Using this method, we show that while erythrocytes of MAFLD patients exhibit sphingosine accumulation, the utilisation of exogenous sphingosine from their erythrocytes is not affected. This suggests that the metabolic shift may be driven by increased sphingosine supply from the plasma.

BackgroundTrauma from occlusion (TFO) is a frequently under-recognized clinical entity. While narrative reviews exist, no prior systematic review has quantitatively synthesized the prevalence of TFO signs in orthodontic patients, the distribution of the Akerly classification for deep traumatic overbite, the efficacy of orthodontic intrusion, or the outcomes of immediate orthodontic repositioning of traumatized incisors. Furthermore, the knowledge-practice gap among orthodontists regarding trauma management has not been meta-analyzed. MethodsSystematic review and meta-analysis of observational and interventional studies, including cross-sectional studies, randomized controlled trials, and before-after studies. We searched PubMed (n=57), PubMed Central (n=538), the Cochrane Library (n=11: 2 reviews, 9 trials), and Google Scholar (~3,930) up to December 2025. Studies reporting prevalence of TFO signs, Akerly classification distribution, overbite reduction following orthodontic intrusion, success of immediate orthodontic repositioning, or orthodontist knowledge/practice were included. Random-effects meta-analyses were performed using the meta package in R (DerSimonian-Laird estimation for {tau}2). The protocol was not registered due to the exploratory nature of this multi-domain synthesis; however, the methodology strictly adhered to PRISMA 2020 guidelines. ResultsTwenty-seven studies (n=8,432 participants) were included. The pooled prevalence of any TFO sign was 34% (95%CI:27-42%, I2=86%), with wide prediction intervals indicating substantial between-study variability. TFO was variably defined across studies as the presence of [&ge;]1 of the following: fremitus, increased mobility, occlusal interference, soft tissue trauma, or CR-CO discrepancy. Higher prevalence was observed in Class II malocclusion (46% vs. 22%). Among deep traumatic overbite cases classified using the Akerly system, Type II was most common (52%, 95%CI:44-60%), followed by Type I (31%) and Type III (17%). Orthodontic intrusion reduced overbite by a mean of 2.8 mm (95%CI:2.1-3.5, I2=72%); TAD-assisted intrusion produced greater reduction (3.4 mm) than conventional archwires (2.1 mm, p<0.001). Immediate orthodontic repositioning of traumatized incisors with light forces ([&le;]50 g) achieved 91% success (95%CI:84-96%) at 12 months, comparable to splinting (84%), with no statistically significant difference between groups. The orthodontic group required fewer visits and reported better comfort. Meta-analysis of orthodontist knowledge showed correct awareness of specific trauma management protocols was below 40% in most domains, indicating a substantial evidence-practice gap. ConclusionThis first systematic review and meta-analysis on TFO in orthodontics provides preliminary quantitative benchmarks. One-third of orthodontic patients exhibit TFO signs; Akerly Type II is the dominant deep overbite pattern; orthodontic intrusion effectively reduces overbite by approximately 3 mm; immediate light-force repositioning is comparable to splinting in success and superior in efficiency. However, the disconnect between high clinical efficacy (e.g., 91% success of repositioning) and low practitioner awareness (<40%) represents a substantial translational gap in clinical practice. Assessment of publication bias was limited due to the small number of studies in several analyses (<10), precluding reliable funnel plot interpretation.

BackgroundWhite spot lesions (WSLs) affect up to 95% of patients after fixed orthodontic treatment. These demineralized areas harm aesthetics and may become more visible after tooth bleaching. Resin infiltration offers a micro-invasive masking technique. ObjectiveTo systematically review and meta-analyze the efficacy of resin infiltration for masking post-orthodontic white spot lesions compared with no treatment, placebo, or alternative remineralizing agents. MethodsWe followed PRISMA 2020 guidelines. We searched electronic databases (PubMed Central, Google Scholar, CORE, Epistemonikos, DOAJ) from inception to April 24, 2026, using database-specific search strings. We included randomized controlled trials (RCTs) and prospective clinical studies that evaluated resin infiltration for post-orthodontic WSLs in human participants. The primary outcome was change in lesion visibility. Two authors assessed risk of bias using Cochrane ROB-2 (RCTs) and ROBINS-I (non-randomized studies). We performed a random-effects meta-analysis using R (version 4.3.1; meta package) and estimated between-study variance ({tau}2) with the DerSimonian-Laird method. ResultsTen studies (6 RCTs, 4 prospective cohorts) with 1,204 patients and 3,847 WSLs met the inclusion criteria. Resin infiltration significantly reduced lesion visibility compared with no treatment (standardized mean difference [SMD] = -1.78; 95% CI: -2.24 to -1.32; p < 0.001; I2 = 65%) and compared with fluoride varnish (SMD = -1.42; 95% CI: -1.82 to -1.02; p < 0.001; I2 = 48%). The effect remained stable at 12-24 months. Patient satisfaction ranged from 84% to 94%. Mild transient sensitivity (11%) was the only reported adverse event. Funnel plot inspection showed no obvious small-study effects. ConclusionsResin infiltration shows high efficacy and durability for masking post-orthodontic white spot lesions, with a very large effect size. Clinicians should consider it the first-line minimally invasive aesthetic treatment before any tooth whitening procedure.

Heat shock protein 90 (HSP90) assists protein folding and maturation of many important signaling proteins. In various diseases, HSP90 clients contribute to aberrant signaling, and HSP90 inhibition is being explored as a potential therapeutic approach. Commonly researched HSP90 inhibitors target the ATP-binding pocket, thereby disrupting the ATP-induced closure of HSP90. Drugs disrupting the HSP90 ATPase cycle by targeting the closed state of the chaperone have not been developed. Here, we present de novo design and selection of protein binders interacting exclusively with the closed state HSP90. Two such binders, H2 and H4, were identified that feature a similar fold and comparable affinities for HSP90 but display different binding kinetics. The structures of the HSP90 complexes with H2 and H4 were determined by cryo-EM single particle analysis, and they revealed high accuracy of the BindCraft predictions. H2 and H4 compete with p23 at one but not both symmetrical p23 binding sites on HSP90. H2 expressed in HEK293T cells moderately elevated expression of HSP70 and had no effect on the HSP90 level, suggesting muted heat shock response. Overall, this study demonstrates that the de novo binders represent novel and promising tools to probe the potential utility of HSP90 inhibition by targeting its closed state.

Early postnatal development is a critical period for hippocampal circuit maturation. While postnatal hippocampal development has been mostly studied in rats, less is known about the developmental trajectory of electrophysiological properties in mice, despite the wide use of these animal models for molecular and genetic studies of nervous system. In this study, we investigated the postnatal maturation of hippocampal CA1 pyramidal neurons in male and female wild-type mice. Whole-cell patch-clamp recordings were performed in acute hippocampal slices to assess passive and active membrane properties as well as spontaneous excitatory synaptic activity. We found that maturation of neuronal firing properties was associated with faster responses to stimulation, higher-amplitude and shorter-duration action potentials, and more precise control of neuronal firing. Simultaneously, synaptic activity changed across development, with decreased sEPSC inter-event intervals and stable event amplitudes, suggesting enhanced functional connectivity without major changes in synaptic strength. Sex-dependent differences in electrophysiological properties were observed primarily during the first postnatal week, indicating that sex influences the early trajectory of neuronal maturation. Together, our findings provide a comprehensive electrophysiological baseline for mouse hippocampal CA1 pyramidal neurons during postnatal development.

Microtubules are dynamic filaments of tubulin heterodimers that comprise an essential part of the eukaryotic cytoskeleton1. The nucleotide state of tubulin controls microtubule dynamics: stable GTP-microtubules favor polymerization, whereas unstable GDP-microtubules drive depolymerization2. Anticancer compounds such as Taxol (paclitaxel) target microtubule dynamicity by preventing microtubule depolymerization3,4. Despite decades of work, the molecular basis of microtubule dynamics remains poorly defined. Using cryo-EM, we determined [~]2.2 [A] structures of human microtubules in GTP-like (GMPCPP) and GDP states. Comparison of these two states revealed switch-like structural changes as tubulins transition from the pre-hydrolysis (GMPCPP) to the post-hydrolysis (GDP) state. Additional structure determination of Taxol-bound microtubules at [~]2.2 [A] showed that Taxol binding converts the microtubule lattice into a pre-hydrolysis state by reversing the structural switches flipped during GTP hydrolysis. Focusing our analysis on the microtubule seam shows that the pre-hydrolysis conformation of GMPCPP or Taxol-GDP exhibits favorable lateral interactions at the seam, with lattice deformations clearly visible at the GDP seam. Together, our data show the existence of structural switches in tubulin that are coupled to the nucleotide state and are exploited by Taxol to stabilize microtubules into a pre-hydrolysis-like state. (191 words)

Careful balance of the redox status of the embryo and reduction of oxidative stress is crucial in early development. Here we show that the culture of preimplantation mouse embryos in the conditionally non-essential amino acid L-proline (Pro) increases the intracellular concentration of the potent antioxidant glutathione as shown by staining of 2-cell, 4-cell and 8-cell embryos with tetrafluoroterephthalonitrile (4F-2CN). Further, liquid-chromatography/mass spectrometry showed increased GSH levels in all Pro-treated preimplantation stages of development compared to controls. The GSH:GSSG ratio also showed a Pro-dependent increase. Overall, our results indicate that the beneficial effect of Pro in preimplantation embryo culture is due to the reduction in oxidative stress mediated through an increase in cellular GSH concentration.

Background: Endothelial progenitor cells (EPCs) contribute to endothelial repair and neovascularization, and EPC dysfunction is closely associated with oxidative stress-related vascular injury. Forkhead box O3a (FoxO3a) regulates cellular stress responses, whereas miR-34a has been implicated in endothelial dysfunction, senescence, and apoptosis. However, the relationship between FoxO3a and miR-34a-3p in oxidatively injured EPCs remains incompletely defined. Objective: This study investigated the role of FoxO3a in H2O2-induced EPC dysfunction and examined whether miR-34a-3p directly interacts with the FoxO3a 3' untranslated region (3'UTR). Methods: Human umbilical cord blood-derived EPCs were identified by DiI-ac-LDL uptake, FITC-UEA-1 binding, and the expression of EPC-related markers. Oxidative stress was induced by H2O2. Cell viability, apoptosis, and angiogenic capacity were evaluated using CCK-8 assay, Annexin V/7-AAD flow cytometry, and Matrigel tube formation assay, respectively. FoxO3a expression was modulated using adenoviral overexpression or knockdown vectors, and miR-34a was modulated using mimics or antagomir. FoxO3a and miR-34a expression levels were detected by Western blot and qPCR. A dual-luciferase reporter assay was used to verify the interaction between hsa-miR-34a-3p and the FoxO3a 3'UTR. Results: H2O2 reduced EPC viability, increased apoptosis, and impaired tube formation in a concentration-dependent manner. H2O2 increased FoxO3a protein abundance and miR-34a expression, whereas FoxO3a mRNA did not change markedly. FoxO3a overexpression aggravated, whereas FoxO3a knockdown partially alleviated, H2O2-induced EPC dysfunction. Similarly, miR-34a mimics further suppressed EPC viability and tube formation, while miR-34a antagomir exerted a protective effect. Dual-luciferase reporter analysis showed that hsa-miR-34a-3p significantly reduced the activity of the wild-type FoxO3a 3'UTR reporter, while mutation of the predicted binding site abolished this suppression. Conclusion: FoxO3a and miR-34a participate in oxidative stress-induced EPC dysfunction. The dual-luciferase data demonstrate that hsa-miR-34a-3p directly targets the FoxO3a 3'UTR, suggesting the presence of miR-34a-3p-mediated post-transcriptional feedback within the FoxO3a-related stress-response network in EPCs.

New approach methodologies (NAMs), including induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) and retinal pigment epithelium (iRPE), are increasingly applied to study retinal disease mechanisms and therapeutic strategies. However, these models often remain relatively immature. Given the high lipid content and complex metabolism of the retina, it is unclear to what extent iPSC-derived systems recapitulate the human retinal lipidome. Here, we compared the lipidomic profiles of ROs and iRPE, collected at several differentiation stages, with those of post-mortem adult human macular, non-macular and RPE plus choroid (pmRPE). The lipidome of iRPE differed markedly from pmRPE, whereas prolonged differentiation of ROs resulted in a lipidomic profile increasingly resembling that of the post-mortem retina. Moreover, ROs showed similarities to both macular and non-macular lipidome. These findings show that iPSC-derived models can become valuable NAMs to study lipid-related retinal disorders and provide a framework to optimize differentiation protocols.

Aim: Cross-sectional summaries of periodontitis based on clinical attachment loss (CAL) are, by definition, conditioned on surviving teeth. Because the most severely affected teeth are more likely to have been lost, these measures may underestimate cumulative disease burden and show an artificial flattening (attenuation) of severity with age. We hypothesised that measures more sensitive to severe attachment loss would show greater attenuation at older ages than measures defined across a broader range of sites. Materials and Methods: Using nationally representative data from adults aged 30+ years in NHANES 2009-2014, we examined age-specific trajectories across multiple continuous measures of periodontal severity and assessed whether divergence between measures followed the pattern predicted under severity-dependent tooth loss. Results: The proportion of observable sites declined from 93% at ages 30-34 to 68% at 80+ years, establishing the structural basis for the divergence observed across severity measures. All severity measures showed nonlinear attenuation with age, with distortion increasing with severity threshold. Higher-threshold measures exhibited the greatest attenuation, while lower-threshold measures showed more stable trajectories. Conclusions: Cross-sectional summaries of periodontitis reflect disease among surviving teeth rather than cumulative damage across teeth originally at risk. Attenuation at older ages is consistent with depletion of the most severely affected teeth rather than biological slowing. Distortion varies by measure, with higher-threshold and mean-based indices most affected, whereas the CAL 3+ mm threshold provides a more stable basis for age comparisons.

BackgroundFriction at the bracket-archwire interface is traditionally considered a key determinant of orthodontic tooth movement efficiency. However, clinical evidence remains inconsistent despite advances in low-friction systems, including self-ligating brackets, coated archwires, and frictionless mechanics. ObjectiveTo evaluate the clinical impact of friction-related interventions on tooth movement, anchorage control, and patient-centered outcomes. MethodsA scoping review with supplementary meta-analysis was conducted following PRISMA-ScR guidelines. Electronic searches of the Cochrane Library (1 systematic review: CD003453), PubMed (128 primary studies), and Google Scholar (approximately 2,500 results, screened to 45 relevant studies) were performed in February 2026 . Randomized controlled trials comparing friction-modifying interventions were included. Primary outcomes included rate of tooth movement, anchorage loss, and molar rotation. Secondary outcomes included pain and treatment duration. Random-effects meta-analysis (DerSimonian-Laird method) was performed using RevMan 5.4; this method was chosen due to expected clinical heterogeneity . Heterogeneity was assessed using the I{superscript 2} statistic and classified using non-overlapping thresholds: 0-40% low, 40-60% moderate, 60-90% substantial, and 90-100% considerable heterogeneity. Risk of bias was assessed using Cochrane RoB 2, and certainty of evidence was evaluated using GRADE. Given the small number of studies, pooled estimates should be interpreted cautiously due to potential small-study effects. ResultsNineteen RCTs were included in quantitative synthesis. Frictionless mechanics did not significantly increase the rate of space closure (MD = 0.15 mm/month; 95% CI: -0.08 to 0.38; P = 0.20; I{superscript 2} = 68% [substantial heterogeneity]) but resulted in significantly greater molar rotation (MD = 6.1 degrees; 95% CI: 4.8 to 7.4; P < 0.001; I{superscript 2} = 45% [moderate heterogeneity]) . Self-ligating brackets showed no consistent advantage in treatment duration or pain reduction. Active self-ligating brackets demonstrated slightly faster alignment than passive systems (MD = 10.24 days; 95% CI: 2.80 to 17.68). Low-friction ligatures and coated archwires did not improve clinical efficiency. Surgical acceleration methods reduced treatment time by 25-50% but increased early discomfort. Low-level laser therapy showed potential for accelerating tooth movement and reducing pain. ConclusionsHigh-level clinical evidence does not support the long-held assumption that reducing friction accelerates orthodontic tooth movement. The evidence fails to demonstrate a clinically meaningful acceleration effect from friction reduction alone. Resistance to sliding appears to be predominantly governed by binding and biological patient response, not friction alone--necessitating a shift in biomechanical strategy. A proposed evidence-informed conceptual model and clinical algorithm are presented to guide decision-making.

Background: Periodontitis is defined by cumulative, irreversible tissue destruction, yet population-based measurement typically relies on cross-sectional indicators derived from retained teeth. Destruction that occurred earlier in life, particularly disease severe enough to result in tooth loss, is structurally excluded from these measures, potentially leading to systematic underestimation of lifetime periodontal burden. Objective: To develop and evaluate a measurement framework that estimates lifetime periodontal burden from cross-sectional data by explicitly incorporating informative tooth loss under etiological uncertainty. Methods: Data were drawn from 10,324 adults aged [&ge;]30 years participating in the 20090-2016 National Health and Nutrition Examination Survey (NHANES) who completed full-mouth periodontal examination and glycated hemoglobin (HbA1c) testing. Lifetime periodontal burden was estimated by combining observed clinical attachment loss in retained teeth with probabilistic contributions from missing teeth, using three alternative age-stratified attribution schedules derived from epidemiological studies of periodontal extraction. Performance was compared with conventional measures of periodontal severity and extent using distributional analyses, correlations with HbA1c, discrimination of diabetes status, and relative importance analysis. Age-adjusted models were treated as sensitivity analyses. Results: Estimated lifetime periodontal burden exhibited strong, monotonic age gradients across glycemic categories, in contrast to more attenuated patterns observed for severity and extent. Across attribution schedules, lifetime burden showed stronger correlations with HbA1c ({rho} = 0.30-0.32) than conventional measures. In multivariable models including all indices, lifetime burden retained an independent association with HbA1c, whereas severity and extent contributed little unique information. Discriminative performance for diabetes status was consistently higher for lifetime burden than for conventional measures and remained stable across attribution schedules. Conclusions: Lifetime periodontal burden can be estimated from cross-sectional data by explicitly modelling informative tooth loss rather than restricting measurement to retained teeth. Incorporating historical tissue loss under uncertainty yields a more coherent representation of cumulative periodontal destruction than snapshot-based measures and provides a methodological basis for life-course-oriented periodontal epidemiology.

Background: Accurately identifying patients who will require opioids after third molar extraction could improve pain management while supporting opioid stewardship. This study evaluated surgeon accuracy in predicting supplemental opioid use following treatment with ibuprofen and acetaminophen. Methods: Patients (N=85) undergoing third molar extraction were treated with a standardized analgesic regimen of ibuprofen+acetaminophen, with supplemental opioid if needed. Four surgeons independently reviewed preoperative radiographs, assessed surgical difficulty using the Pederson scale, and rated the likelihood of supplemental opioid use on a 5-point Likert scale. Inter-rater reliability was assessed using intraclass correlation coefficients (ICC). The relationship between surgeon ratings and postoperative opioid use was evaluated using logistic regression and receiver operating characteristic (ROC) analysis. Results: Seventeen patients used supplemental opioid analgesics. Inter-rater reliability among surgeons was moderate (ICC3=0.606, 95%CI: 0.505-0.700), while reliability of the average rating across surgeons was good (ICC3k = 0.860, 95% CI: 0.804-0.903). Median surgeon rating was not associated with postoperative opioid use (OR: 0.800, 95% CI: 0.414-1.51, p=0.496) and demonstrated poor discrimination (AUC: 0.551, 95% CI: 0.392-0.710). Surgeon ratings were positively associated with Pederson score (beta=0.073, 95%CI: 0.050-0.096; p<0.001). Conclusions: Surgeons demonstrated moderate agreement, but these assessments did not accurately identify patients who ultimately required supplemental opioids. Surgeon judgments appeared to be influenced by anticipated surgical difficulty. Practical Implications: Clinicians should follow current recommendations against routine "just-in-case" opioid prescribing after third molar extraction. Future studies should focus on identifying clinical and biological predictors of inadequate analgesic response to NSAIDs to support individualized pain management strategies.

Phenylketonuria (PKU) is a genetic metabolic disorder caused by the lack of functional phenylalanine hydroxylase (PAH). Elevated levels of phenylalanine (Phe) are known to be neurotoxic; however, the molecular mechanisms underlying Phe's effects remain elusive. This study investigates the impact of PKU on proteostasis, redox balance, and metabolism in BTBR PAHenu2 mice, a severe disease animal model. Combined proteomics and metabolomics revealed impaired redox homeostasis in the brain and disrupted mitochondrial energy metabolism (ATP and TCA intermediates). The dysregulation was further supported by decreased levels of ATP, reduced glutathione (GSH), cysteine, and reduced catalase activity. Western blot analyses revealed substantial remodeling of protein degradation systems: the 19S regulatory (Rpt1) subunit and 26S proteasome content and activity were significantly increased, and ubiquitinated protein levels were elevated, indicating protein turnover and activation of the ubiquitin-proteasome system. Autophagy was also activated, as evidenced by a reduced LC3-II/LC3-I ratio, decreased p62 levels, unchanged ATG5 levels, and increased HSPA8 protein expression. By contrast, UPR markers remained stable despite an increase in the oxidized-to-reduced PDI ratio, suggesting a localized shift without activation of a full ER stress response. In parallel, systemic alterations were assessed in whole blood. Indeed, GSH, cysteine, ATP and ADP were decreased in PKU, whereas NADPH increased. These changes were accompanied by reduced activities of GSH reductase and GSH peroxidase, thereby confirming metabolic and redox disruption. Collectively, these findings indicate that PKU is associated with activation of protein degradation pathways as an adaptive response to cellular stress combined with redox imbalance and energy dysregulation.

Cell replacement represents a potential treatment modality for retinal disorders characterized by photoreceptor loss. However, photoreceptor replacement approaches have not been clinically established. To take this forward, the main goal of this study was to systematically compare human photoreceptors of different ages and identify those that enable functional integration into the degenerative retina. Donor cells were isolated from iPSC-derived retinal organoids generated by a GMP-compliant protocol at differentiation days 120, 150, or 200 and transplanted subretinally into cone photoreceptor function loss 1 (Cpfl1) recipients, an inherited mouse model of cone degeneration. While younger photoreceptors showed slightly improved transplantation outcomes, donor photoreceptors of all culture stages displayed long-term survival, cone identity, structural integration into the host retina, and tight interactions with host Mueller glia, including formation of a continuous outer limiting membrane. Transplanted photoreceptors showed signs of advanced maturation, including correct polarization with generation of apical inner- and outer segments, while basal synapses were formed with host bipolar cells. Electrophysiological assessment of host retinal ganglion cells revealed light-evoked responses in transplant-containing regions, providing evidence for functional incorporation of human photoreceptors into the mouse neuro-retinal circuitry. Thus, GMP-compliant human iPSC-derived photoreceptors are stable over a wide range of differentiation stages and constitute a robust cell source for retinal transplantation and functional repair. The findings provide important prerequisites for the development of standardized procedures towards clinical translation of photoreceptor replacement in the retina.

Retinopathy of prematurity (ROP) is caused by hyperoxic exposure of prematurely born infants. The mouse model of oxygen-induced retinopathy (OIR) recapitulates pathological features of both phase I and phase II ROP. We here looked at the retinal proteins that change in response to hyperoxia in phase I of the mouse model of OIR. Using tandem mass tag labeled proteomics, we found several differentially expressed proteins (DEPs) in phase I of OIR. Of all the DEPs, we investigated the role of previously unknown protein NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2). NDUFA4L2 protein and its paralog NDUFA4 are both mitochondrial complex I proteins; however, here we demonstrate that NDUFA4L2 changes in both phases of OIR, with no changes in its paralog NDUFA4, implying its unique function in pathophysiology of the disease. We demonstrate that NDUFA4L2 is an oxygen-sensitive protein and regulates retinal endothelial cell migration by rescuing isocitrate dehydrogenase flux impaired by hyperoxia in phase I of OIR.

Glioblastoma (GBM) is the most aggressive primary brain malignancy, characterized by hypoxia-driven proliferation, therapeutic resistance, and poor prognosis. While hypoxia-induced transcriptional changes are well documented, the temporal regulation of cell cycle genes under sustained hypoxia remains unclear. This study profiled transcriptomic alterations in U87MG cells cultured under normoxia and graded hypoxia for one to three days. Differentially expressed genes (DEGs) were identified and analyzed using STRING, Cytoscape, MCODE, and CytoHubba to construct protein-protein interaction (PPI) networks and extract hub genes. Functional enrichment was assessed through DAVID, ClueGO, and KEGG, while prognostic relevance was evaluated using GlioVis and ONCOMINE datasets. qRT-PCR validated expression of selected hub genes. A total of 294 DEGs were identified, forming two main functional modules enriched in cell cycle regulation and chemokine signaling pathways. Eighteen hub genes (KIF20A, CCNB1, AURKA, EGR1, CDCA3, CENPF, CDCA2, ASPM, KIF11, CCL2, CCNA2, DLGAP5, RACGAP1, TPX2, PTGS2, CTGF, and KIFC1) were significantly associated with mitotic processes and GBM progression. Survival analysis demonstrated that 17 of these genes correlated with poor overall survival (p < 0.05). qRT-PCR confirmed that hub gene expression peaked during early hypoxia and declined with prolonged exposure, indicating dynamic regulatory adaptation. These findings identify key hypoxia-responsive genes governing cell cycle progression and highlight their prognostic and therapeutic potential in glioblastoma.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.