Biomolecules

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.

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.

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.

Emerin (EMD), an inner nuclear membrane protein essential for nuclear architecture integrity, gene expression, cellular signaling, and chromatin stability, interacts with the LINC complex and participates in cytoskeleton-nucleoskeleton communication by binding to nuclear actin filaments. EMD is implicated in migration, invasion, and metastasis in some tumors, but its role in glioblastoma (GBM) remains unclear. This study evaluated the effects of EMD knockdown and overexpression in GBM cell lines following genotoxic treatment with cisplatin. In both wild-type p53 (U87-MG) and mutant p53 (U138-MG) GBM cells, EMD expression is high, and cisplatin treatment did not affect these protein levels. EMD knockdown in U87-MG cells significantly increased cisplatin IC50, viability, and proliferation. Conversely, stable overexpression of EMD in U87-MG cells led to reduced cisplatin IC50, viability, proliferation, and migration. EMD knockdown or overexpression did not affect any U138-MG phenotypes, with or without cisplatin treatment. Modulation of EMD levels causes morphological changes in stress fiber cytoskeleton, whereas overexpression of EMD in U87-MG cells promotes an increase and a decrease in nuclear and cytoplasmic actin levels, respectively. These biological responses of U87-MG cells overexpressing EMD were coincidentally associated with alterations in the levels of pH2AX(Ser139), p-p53(Ser15), p53, and p21Kip1 proteins after cisplatin exposure. In sum, modulation of EMD levels affects the viability, migration, and proliferation of wild-type p53 GBM cells treated with cisplatin, suggesting unknown roles in the DNA damage response and repair. This work highlights EMD as a potential regulator of GBM chemoresistance and a target for therapeutic intervention.

Background: Chronic and non-healing wounds remain a major clinical challenge with limited therapeutic options. Angiogenesis and inflammation are central to tissue repair, and mesenchymal stem cells (MSC) contribute to these processes through their trophic and immunomodulatory secretome. Cannabidiol (CBD) exhibits antioxidant and immunomodulatory properties. However, whether CBD-rich Cannabis sativa extract stimulate MSC toward a pro-angiogenic secretome remains unclear. Purpose: This study aims to determine whether purified CBD or a phytochemically CBD-rich full spectrum extract stimulate umbilical cord-derived human MSC (UC-hMSC) to secrete pro-angiogenic factors and enhance endothelial responses relevant to wound healing. Methods: UC-hMSC were preconditioned with either purified CBD or a CBD-rich full-spectrum extract. Transcriptional changes were assessed by qPCR. The functional impact of the resulting secretome was evaluated in vitro using HUVEC-based proliferation and tube formation assays, and in vivo through the chick chorioallantoic membrane assay. To explore underlying mechanisms, we examined HIF-1 stabilization and VEGFA release in UC-hMSC, and VEGFR-2/ERK signaling in HUVEC. Results: Purified CBD and full-spectrum CBD extract preconditioned UC-hMSC secretomes, increased HUVEC proliferation, tube formation, and enhanced vascular branching in the CAM assay. Mechanistic analyses indicated activation of the HIF-1/VEGF axis in UC-hMSC, and ERK1/2 activation in HUVEC that was sensitive to VEGFR-2 blockade. Conclusion: Purified CBD and CBD-rich full-spectrum extract prime UC-hMSC toward a pro-angiogenic secretome that promotes endothelial activation and neovascularization. These findings suggest that cannabinoid-based preconditioning of UC-hMSC involves the HIF-1/VEGF axis and VEGFR-2/ERK signaling pathways in endothelial cells, supporting further investigation of this approach in wound healing and regenerative therapies.

BACKGROUND: Anemia is a negative factor in cancer, influencing the prognosis, quality of life and financial situation of cancer patients. Recent studies have shown that anemia in cancer is provoked by augmented erythrocyte removal. OBJECTIVE: In this study we sought to investigate the molecular bases for erythrocyte removal in cancer patients with anemia. In particular, we explored the levels of erythrocyte CD47, lactadherin, calreticulin and MCP1. METHODS: Thirty five anemic cancer patients (25 women, aged 66.4 +/-11.35 years old) and twelve healthy non-anemic controls (8 men, aged 61.1+/-9.98 years old) participated in our study. Red blood cells were isolated throug multiple centrifugations, and were lysed with the use of Triton-X 100. The levels of CD47, lactadherin, calreticulin and monocyte chemoattrractant protein 1 were determined by ELISA. RESULTS: Erythrocytes of anemic cancer patients display reduced CD47 (p<0.001), MCP1 (p=0.05), CD47 to lactadherin ratio (p<0.05), and increased lactadherin levels (p<0.01) in comparison to the healthy controls. DISCUSSION: Reduced CD47 along with increased lactadherin possibly drive erythrocyte removal in anemic cancer patients. Conclusions: The role of CD47 and increased lactadherin should be examined in the future as potential therapeutic targets and biomarkers for anemia diagnosis and iron dymsetabolism.

Background: Uncontrolled bleeding complicates trauma, surgery and many medical conditions. While currently available procoagulant therapies (e.g., plasma-derived factors, recombinant proteins, antifibrinolytics) have crucial limitations. Methods: N389 (CQQTVTLLPAADLDDFSC) was synthesized by Fmoc solid-phase chemistry, characterized by HPLC and LC-MS, then tested in normal human pooled plasma in microplate mechanical clot-formation assays using incubated and immediate addition formats. Kinetic parameters (plasma recalcification, PRT; maximum absorbance, Amax) were obtained from absorbance curves fit to four-parameter logistic models. Mixing studies with modified (i.e., aged, adsorbed) plasma probed factor dependence. Results: In plasma coagulation assays activated with 25 mM CaCl2, baseline clotting showed a PRT of 23.74 +/- 0.27 min and Amax of 0.1813 +/- 0.0043 (n = 3), whereas N389 significantly reduced PRT to 8.442 +/- 6.0395 min without incubation (p = 0.0012), further decreased PRT after incubation (p < 0.0001), increased Amax to 0.2523, and retained comparable activity across normal, adsorbed, and aged plasma, in contrast to S1255 which showed a faster but incubation-labile effect with PRT 2.353 +/- 1.3685 min (p = 0.0007) and marked attenuation in factor-depleted and aged plasma. Mixing studies showed N389 activity persisted across normal, aged and adsorbed plasma, consistent with a mechanism that does not require intact plasma coagulation factor profiles (specifically factors II, V, VIII, VII, IX, X). Discussion: Collectively with prior evidence on anionic surfaces, Ca2+-binding Gla domains, and peptide-modulated fibrin polymerization, these results support a model in which N389 functions as a stable, charge-based scaffold that coordinates divalent cations and/or directly nucleates fibrin(ogen), while highlighting limitations of bulk clotting assays and the need for targeted thrombin generation, binding, aggregation, and contact-activation studies. Conclusions: The aspartate-rich peptide N389 is a sustained, factor-independent procoagulant at least in vitro. N389 thus merits further mechanistic and translational evaluation as a synthetic hemostatic agent.

BackgroundPrenatal cannabis use is becoming increasingly more commonplace. However, cannabis exposure is linked to adverse pregnancy outcomes, including gestational hypertension, preeclampsia, and preterm birth. The aim of this study was to determine the morphological and molecular effects of prenatal cannabinoid exposure on the placenta. MethodsPregnant Sprague-Dawley rats were exposed daily to vaporized THC (100 mg/mL) starting at gestational day (GD)5 until GD19 when dams were sacrificed and fetuses and placentas collected. Fetuses were genotyped for genetic sex and transcriptomic analysis was performed on male and female THC-exposed and control placentas. ResultsOn GD19, both the fetuses and placentas from the THC group were significantly larger than the control. When separated by sex, both male and female THC fetuses were significantly larger; however, only male THC placentas were significantly larger than male control placentas with no significant difference in placental weight between female control and THC placentas. RNA-sequencing revealed enriched biological processes related to nutrient transport and lipid catabolism, protein-lipid complex formation, and lipoprotein particle remodeling and organization. Further transcriptomic analysis determined that the differentially expressed genes and enriched biological processes related to lipid metabolism were preferentially enriched in the female THC placentas compared to the male, suggesting a sex-specific effect. DiscussionCollectively, these data present sex-specific effects of prenatal cannabinoid exposure on placental growth and global gene expression. These data also suggest that sex influences gene expression of genes related to lipid metabolism in the THC-exposed placentas.

Relapse of aligned mandibular anterior teeth and the progressive collapse of the mandibular anterior arch are historically striking problems for orthodontists. The etiology of this collapse, and the cause of mesial molar drift, are unknown. However, light continuous (quasi-continuous) intra-oral pressures and forces applied to the mandibular dentition have been implicated. To explore this further, we use three-dimensional finite element analysis to investigate the influence of these intra-oral loads (tongue pressure, lip-cheek pressure, and interdental force) on mandibular arch collapse and mesial molar drift. Dentitions of three-dimensional finite element mandibular models were subjected to a wide range of simulated tongue pressures, lip-cheek pressures, and transseptal fiber-mediated interdental forces reported in the literature. Resulting crown displacement measurements from these isolated loads were made along with measurements resulting from simultaneous combined application of literature-defined mean tongue pressure, lip-cheek pressure, and interdental force. Our results indicate that tongue pressure alone results in generalized arch expansion and tooth spacing while lip-cheek pressure and interdental force result in generalized arch collapse, anterior crowding, and mesial molar displacement. Simultaneous application of tongue pressure, lip-cheek pressure, and interdental force mean values, as would occur in vivo, results in incisor crowding, intercanine width reduction, and mesial molar displacement. Our results suggest mandibular anterior arch collapse (incisor crowding / intercanine width reduction), and mesial molar displacement result from simultaneous application of tongue pressure, lip-cheek pressure, and interdental force.

Calreticulin mutations in myeloproliferative neoplasms result in the replacement of the C-terminus acidic sequence with a positively charged tail that causes pathological activation of the thrombopoietin. The two canonical variants are Type-1 and Type-2. The remaining are mainly classified as Type-1 or Type-2 like based on the wild type sequence retained. Here, we performed in silico biophysicochemical analyses of 76 CALR exon 9 frameshift variants by their sequence and predicted biophysical properties, complemented by structural modeling of the mutant homodimers. Beyond confirming the Type-1 versus Type-2 distinction, we found that the Type 1-like variants form a continuum of charge architecture along which two reproducible subgroups can be identified, rather than sharply separated classes. This work refines the conventional mechanism-based classification into a charge-resolved framework and provides testable hypotheses linking novel-tail chemistry to receptor activation in CALR-mutant neoplasms and paves the way for improved targeted therapies based on individual mutants characteristics

Purpose: Photoreceptor Kv2.1/Kv8.2 voltage-gated potassium channels carry an outward potassium current, helping to set the resting membrane potential and to shape dim light responses. Because potassium flux in the outer retina influences extracellular osmolarity and fluid distribution, we hypothesized that Kv2.1/Kv8.2 channels also contribute to fluid homeostasis in this region of the retina. Methods: OCT imaging was performed in Kv8.2 heterozygous (Het) and knockout (KO) mice aged 4-7 weeks under dark- and light-adapted conditions. Light-dark differences in the distance between the external limiting membrane (ELM) and retinal pigment epithelium (RPE) ({Delta}ELM-RPE) were calculated to quantify light-evoked expansion of the subretinal space (SRS). As a secondary outcome, outer nuclear layer (ONL) thickness was also measured under both lighting conditions. Retinal gene expression was assessed by RNA-seq and droplet digital RT-PCR. Retinal protein expression was determined by western blotting and immunolabeling. Results: {Delta}ELM-RPE was significantly reduced in Kv8.2 KO mice compared with Het controls, indicating reduced SRS hydration. ONL thickness exhibited a small but significant light-dark change that was different between genotypes. Transcriptomic analyses revealed upregulation of osmosensitive genes, including osmolyte transporters and aquaporins. AQP1 protein expression in photoreceptors increased. Conclusions: These findings reveal a previously unrecognized role for Kv2.1/Kv8.2 channels in outer retinal fluid homeostasis and support a model in which photoreceptor potassium efflux contributes to osmotic water movement into the subretinal space.

Mechanotransduction via the actin cytoskeleton is linked to fundamental cellular processes such as morphogenesis, cell division, and motility, requiring the control of tensile forces mediated by the motor protein non-muscle myosin 2 (NM2). Formins such as mDia1 have been shown to elongate actin structures that are under mechanical tension; conversely, mDia1s elongation rates are modulated by the applied force. Despite their relevance at the membrane/cortex interface, reported values for tension in formin-elongated actin filaments stem from theoretical estimates and simulations, but have not been amenable experimentally so far. Thus, we developed a Forster resonance energy transfer (FRET)-based, tension-sensitive probe (mDia1TS) and quantified the measured tension in live U2OS cells using fluorescence lifetime imaging microscopy (FLIM). Through whole-cell ROI analysis we show a short and long lifetime component, reporting an intensity-weighted, averaged lifetime corresponding to [~]3.5 pN. Upon mitogen stimulation of cells using EGF, we show that the tension homeostasis changed significantly, with a measurable increase in tension in the cells periphery and relaxation in its center. Furthermore, the reported average tension relaxed by 2 pN after adding the NM2 inhibitor para-nitroblebbistatin. We utilized siRNA knockdowns of individual NM2 paralogs (NM2-A, NM2-B, or NM2-C) to measure their individual contribution, revealing NM2-A as the main paralog to produce tensile force in this system. Taken together, we demonstrate that mDia1TS is able to directly determine that active mDia1 in cells is under tension, and that subcellular quantification with pN precision is possible. SignificanceDespite the fundamental importance of formins in regulating actin-based processes, reported values for tension in formin-mediated actin structures stem from simulations and theoretical estimates. In this study we developed a FRET-based, tension-sensitive reporter probe for formin mDia1, which we termed mDia1TS. Given the expanding clinical spectrum of DIAPH1/mDia1 mutations, our tool mDia1TS provides a quantitative tool for elucidation of changes in cytoskeletal assemblies.

Background: Stress-induced childhood-onset neurodegeneration with variable ataxia and seizures (CONDSIAS) is a rare autosomal recessive disorder caused by biallelic variants in ADPRHL2, which encodes ADP-ribosylhydrolase 3 (ARH3), a key enzyme involved in poly (ADP-ribose) (PAR) metabolism. Although Minocycline has been reported to attenuate PAR-mediated neurotoxicity primarily through modulation of PARP-dependent pathways, whether it may also interact with ARH3 or influence the structural behavior of pathogenic ARH3 variants remains unknown. This study was designed to explore this possibility by integrating clinical observation with computational structural analyses. Methods: Comprehensive clinical evaluation, targeted Sanger sequencing, and in silico pathogenicity analyses were performed. Protein modeling, molecular docking, and 100-ns molecular dynamics simulations were conducted to evaluate the predicted structural consequences of the p.Thr79Pro variant and to explore potential interactions between ARH3 and Minocycline. Results: A homozygous ADPRHL2 variant (NM_017825.3:c.235A>C; p.Thr79Pro) was identified in a child with CONDSIAS. Computational analyses predicted reduced structural stability and increased conformational flexibility of the mutant ARH3 protein relative to the wild-type structure. MM-GBSA calculations estimated differences in binding free energies between the wild-type (-34.51 kcal/mol) and mutant (-39.76 kcal/mol) ARH3-Minocycline complexes, suggesting subtle differences in their predicted energetic profiles. Clinically, neurological progression appeared stable, with improved motor function observed during approximately one year of follow-up and no notable treatment-related adverse effects. Conclusions: By integrating clinical observations with computational structural analyses, this study provides preliminary computational support for the hypothesis that Minocycline may influence ARH3 conformational behavior in addition to its proposed effects on PARP-dependent pathways. Although these findings do not demonstrate direct molecular binding or therapeutic efficacy, they provide a biologically plausible framework for future biochemical, cellular, and functional investigations. Keywords: CONDSIAS; ADPRHL2; ARH3; Minocycline; molecular docking; molecular dynamics simulation; structural bioinformatics; translational medicine

Background and AimsAs the older population (aged 65 years and older) continues to expand and more people drink alcohol, aging has been linked to the development of alcohol-associated liver disease (ALD) and to worse disease outcomes. The aim of this study was to explore the mechanisms by which advanced age and alcohol exacerbate alcohol-induced liver injury. MethodsTwo-to-three-month-old and twenty-to-twenty-two-month-old male C57BL/6N mice were subjected to chronic-on-binge alcohol feeding (the Gao-binge model). Serum alanine aminotransferase, aspartate aminotransferase, and triglyceride content were determined using biochemical assays. The levels of lipogenesis, fatty acid-metabolizing proteins, inflammatory markers, mitochondrial and autophagy-related proteins, and senescence-associated proteins were determined by immunoblotting, immunohistochemistry, and real-time quantitative polymerase chain reaction (RT-qPCR). Liver tissues were also subjected to RNA sequencing and metabolomics analyses. Proteomics analysis was performed on serum samples. Tail-vein adenovirus-TFEB was injected to overexpress hepatic TFEB in 22-month-old C57BL/6N male mice, followed by Gao-binge alcohol feeding. ResultsHepatic triglyceride content was significantly increased in aged, alcohol-fed mice, whereas serum ALT and AST levels remained relatively similar between alcohol-fed young and aged mice. Gao-binge alcohol increased the hepatic levels of diacylglycerol and acyl-carnitine species in both aged and young livers. RNA sequencing, proteomic analysis, and serum cytokine array analysis showed that inflammatory cytokines, including Ccr2, Cxcl1, and CCL6, and pro-inflammatory antibody fragments were increased in aged, alcohol-fed mice. Increased gene and protein expression of the senescent markers p21 and p27, along with increased senescent-associated (SA) {beta}-galactosidase activity in ethanol-fed aged mice compared to young mice. Gene and protein expression of TFEB was downregulated in ethanol-fed young and aged animals, along with decreased levels of lysosomal ATPases and hepatic dipeptide content. Overexpression of TFEB in the livers of aged, Gao-binge-fed mice was associated with reduced Ly6G-positive cells, reduced protein levels of the innate immune mediators cGAS, IRF-7, IRF3, and NLRP3, and reduced caspase-1 activity as well as serum ALT levels. ConclusionsOur findings indicate that advanced age perpetuates the detrimental effects of excessive alcohol consumption on various homeostatic processes and promotes steatosis and inflammation in the liver. Modulations in hepatic TFEB could be effective in mitigating pro-inflammatory signaling that occurs due to the synergistic effect of both heavy alcohol consumption and advanced age.

Background Alveolar bone assessment in periodontal practice relies on radiography and clinical probing, both of which have well-documented limitations in precision. Intraoral high-frequency ultrasonography (US) offers a radiation-free alternative with potential for sub-millimeter resolution, the validity and precision for detecting minute osseous changes have not been established. The purpose of this study was to evaluate the concurrent validity and measurement precision of intraoral US for detecting alveolar bone-level changes in patients undergoing crown lengthening and osseous surgery, thereby enabling its translation to monitor osseous changes in patients with periodontitis. Methods Ten patients (28 tooth sites) undergoing crown lengthening or osseous surgery at a USC Advanced Grad Perio clinic were enrolled in this prospective observational study. Distance from the cementoenamel junction (CEJ) to the Alveolar bone crest (ABC) was measured at pre- and post-operative time points using a 40 MHz handheld intraoral US transducer and, intraoperatively, by standardized clinical photography. Agreement was assessed by Pearson correlation and Bland-Altman analysis. Measurement precision was quantified using the standard error of measurement (SEM) and minimum detectable change (MDC). Results Preoperative agreement between methods was excellent (r = 0.977; Bland-Altman bias = -0.009 mm; 95% limits of agreement [LoA]: +-0.40 mm). Post-operative correlation remained strong (r = 0.912; bias = 0.123 mm; LoA: -0.85 to +1.10 mm). Both methods detected statistically significant post-surgical increases in the ABC-to-CEJ distance (p < 0.001), as anticipated. US demonstrated substantially superior precision: preoperative SEM 0.058 mm with US versus 0.128 mm clinically, yielding MDC values of 0.160 mm (US) versus 0.354 mm (clinical), providing a 2.2-fold precision advantage. Conclusions Intraoral US demonstrated strong concurrent validity with clinical photography and a reproducible precision advantage in detecting alveolar bone-level changes in patients with periodontitis. These findings support its clinical utility as a radiation-free, high-sensitivity bone monitoring tool. Larger longitudinal studies with CBCT validation are warranted.

This study compared and evaluated two widely used deep learning-based artificial intelligence (AI) models, U-Net++ and YOLOv11, for quantifying tooth and root exposure on intraoral camera images of the mandibular anterior lingual region. Intraoral images of the mandibular anterior lingual region were collected from 291 patients (mean age, 52.8 years) at a university hospital dental clinic with institutional review board approval (YUMC IRB 2021-07-019-002). A total of 266 eligible images (mean, 5.50 teeth per image; 3.70 teeth with root exposure) were annotated. YOLOv11 and U-Net++ were fine-tuned using five-fold cross-validation with data augmentation. Model performance was evaluated on a held-out test set of 40 images using Dice coefficient, Intersection over Union (IoU), accuracy, mean Average Precision at an IoU threshold of 0.5 (mAP50), Lins concordance correlation coefficient (CCC), and intraclass correlation coefficient (ICC). Confidence intervals were estimated using 10,000 bootstrap iterations. For tooth segmentation, U-Net++ demonstrated superior performance, with high accuracy (0.981), Dice coefficient (0.971), and IoU (0.944). In contrast, for root segmentation, YOLOv11 outperformed U-Net++, achieving higher Dice (0.860 vs. 0.746) and IoU (0.762 vs. 0.631). Notably, YOLOv11 showed stronger agreement with the ground truth for quantifying the exposed root ratio (ERR) (CCC, 0.973; ICC, 0.975). These findings suggest that accurate detection of root exposure is important for assessing periodontal tissue loss and that YOLOv11 is a promising model for root exposure quantification in intraoral images. YOLOv11-based quantification of root exposure may serve as a useful adjunctive AI tool for screening and monitoring periodontal conditions and may support individualized treatment planning.

The development of cost-effective, serum-free media is critical for scalable cultivated meat production. This study used high-throughput screening through a Design of Experiments (DoE) approach to develop an animal-free, serum-free medium (MMM1) specifically for the C2C12 murine myoblasts model cell line with applicability in cultivated meat research including for pet food. Low cost, food-grade inputs such as methylcellulose and spirulina extract resulted in significant cell growth improvements. The optimised MMM1 formulation containing low cost, food-grade inputs, achieved cumulative population doublings comparable to 10% (v/v) fetal bovine serum over four consecutive passages. Furthermore, MMM1 supported scalable cell expansion on commercially available dextran-based microcarriers (Cytodex-3) in both static and agitated conditions in spinner flasks, matching growth rates of serum-based controls. Finally, transitioning to a food-grade DMEM/F12 basal medium maintained cell proliferation equivalent to the pharmaceutical-grade DMEM/F12, but at a significantly lower cost, thus offering a viable strategy to substantially reduce biomanufacturing costs which is a critical challenge in cultivated meat production.