In vivo adenine base editing rescues adrenoleukodystrophy in a humanized mouse model.

X-linked adrenoleukodystrophy (ALD), an inherited neurometabolic disorder caused by mutations in ABCD1, which encodes the peroxisomal ABC transporter, mainly affects the brain, spinal cord, adrenal glands, and testes. In ALD patients, very-long-chain fatty acids (VLCFAs) fail to enter the peroxisome and undergo subsequent ß-oxidation, resulting in their accumulation in the body. It has not been tested whether in vivo base editing or prime editing can be harnessed to ameliorate ALD. We developed a humanized mouse model of ALD by inserting a human cDNA containing the pathogenic variant into the mouse Abcd1 locus. The humanized ALD model showed increased levels of VLCFAs. To correct the mutation, we tested both base editing and prime editing and found that base editing using ABE8e(V106W) could correct the mutation in patient-derived fibroblasts at an efficiency of 7.4%. Adeno-associated virus (AAV)-mediated systemic delivery of NG-ABE8e(V106W) enabled robust correction of the pathogenic variant in the mouse brain (correction efficiency: ∼5.5%), spinal cord (∼5.1%), and adrenal gland (∼2%), leading to a significant reduction in the plasma levels of C26:0/C22:0. This established humanized mouse model and the successful correction of the pathogenic variant using a base editor serve as a significant step toward treating human ALD disease.

Gut microbiota composition and metabolite profiling in smokers: a comparative study between emphysema and asymptomatic individuals with therapeutic implications.

BACKGROUND: Diet has a crucial role in the gut microbiota, and dysbiosis in the gut and lungs has been suggested to be associated with chronic obstructive pulmonary disease. We compared the diet, microbiome and metabolome between asymptomatic smokers and those with emphysema. METHODS: We enrolled 10 asymptomatic smokers with preserved lung function and 16 smokers with emphysema with severe airflow limitation. Dietary intake information was gathered by a self-reported questionnaire. Sputum and faecal samples were collected for microbial and metabolomics analysis. A murine model of emphysema was used to determine the effect of metabolite supplementation. RESULTS: Despite having a similar smoking history with emphysema patients, asymptomatic smokers had higher values of body mass index, fibre intake and faecal acetate level. Linear discriminant analysis identified 17 microbial taxonomic members that were relatively enriched in the faeces of asymptomatic smokers. Analysis of similarity results showed dissimilarity between the two groups (r=0.287, p=0.003). Higher acetate level was positively associated with forced expiratory volume in one second in the emphysema group (r=0.628, p=0.012). Asymptomatic smokers had a greater number of species associated with acetate and propionate (r>0.6) than did those with emphysema (30 vs 19). In an emphysema mouse model, supplementation of acetate and propionate reduced alveolar destruction and the production of proinflammatory cytokines, and propionate decreased the CD3+CD4+IL-17+ T-cell population in the lung and spleen. CONCLUSION: Smokers with emphysema showed differences in diet, microbiome and short-chain fatty acids compared with asymptomatic smokers. Acetate and propionate showed therapeutic effects in a smoking-induced murine model of emphysema.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 directly binds and stabilizes Nrf2 in breast cancer.

Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) has been frequently overexpressed in many types of malignancy, suggesting its oncogenic function. It recognizes phosphorylated serine or threonine (pSer/Thr) of a target protein and isomerizes the adjacent proline (Pro) residue, thereby altering folding, subcellular localization, stability, and function of target proteins. The oncogenic transcription factor, Nrf2 harbors the pSer/Thr-Pro motif. This prompted us to investigate whether Pin1 could bind to Nrf2 and influence its stability and function in the context of implications for breast cancer development and progression. The correlation between Pin1 and Nrf2 in the triple-negative breast cancer cells was validated by RNASeq analysis as well as immunofluorescence staining. Interaction between Pin1 and Nrf2 was assessed by co-immunoprecipitation and an in situ proximity ligation assay. We found that mRNA and protein levels of Pin1 were highly increased in the tumor tissues of triple-negative breast cancer patients and the human breast cancer cell line. Genetic or pharmacologic inhibition of Pin1 enhanced the ubiquitination and degradation of Nrf2. In contrast, the overexpression of Pin1 resulted in the accumulation of Nrf2 in the nucleus, without affecting its transcription. Notably, the phosphorylation of Nrf2 at serine 215, 408, and 577 is essential for its interaction with Pin1. We also identified phosphorylated Ser104 and Thr277 residues in Keap1, a negative regulator of Nrf2, for Pin1 binding. Pin1 plays a role in breast cancer progression through stabilization and constitutive activation of Nrf2 by competing with Keap1 for Nrf2 binding.

A targeted metabolomics approach for sepsis-induced ARDS and its subphenotypes.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is etiologically and clinically a heterogeneous disease. Its diagnostic characteristics and subtype classification, and the application of these features to treatment, have been of considerable interest. Metabolomics is becoming important for identifying ARDS biology and distinguishing its subtypes. This study aimed to identify metabolites that could distinguish sepsis-induced ARDS patients from non-ARDS controls, using a targeted metabolomics approach, and to identify whether sepsis-induced direct and sepsis-induced indirect ARDS are metabolically distinct groups, and if so, confirm their metabolites and associated pathways. METHODS: This study retrospectively analyzed 54 samples of ARDS patients from a sepsis registry that was prospectively collected from March 2011 to February 2018, along with 30 non-ARDS controls. The cohort was divided into direct and indirect ARDS. Metabolite concentrations of five analyte classes (energy metabolism, free fatty acids, amino acids, phospholipids, sphingolipids) were measured using liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry by targeted metabolomics. RESULTS: In total, 186 metabolites were detected. Among them, 102 metabolites could differentiate sepsis-induced ARDS patients from the non-ARDS controls, while 14 metabolites could discriminate sepsis-induced ARDS subphenotypes. Using partial least-squares discriminant analysis, we showed that sepsis-induced ARDS patients were metabolically distinct from the non-ARDS controls. The main distinguishing metabolites were lysophosphatidylethanolamine (lysoPE) plasmalogen, PE plasmalogens, and phosphatidylcholines (PCs). Sepsis-induced direct and indirect ARDS were also metabolically distinct subgroups, with differences in lysoPCs. Glycerophospholipid and sphingolipid metabolism were the most significant metabolic pathways involved in sepsis-induced ARDS biology and in sepsis-induced direct/indirect ARDS, respectively. CONCLUSION: Our study demonstrated a marked difference in metabolic patterns between sepsis-induced ARDS patients and non-ARDS controls, and between sepsis-induced direct and indirect ARDS subpheonotypes. The identified metabolites and pathways can provide clues relevant to the diagnosis and treatment of individuals with ARDS.

Internal structural analysis of the nasomaxillary complex in patients with skeletal class III asymmetry: A study on asymmetry patterns.

OBJECTIVES: To investigate the internal structure of the nasomaxillary complex, including the maxillary sinus, nasal cavity and nasal septum according to the facial asymmetry pattern and to evaluate its correlation with external maxillomandibular asymmetry in Class III patients based on cone-beam computerized tomography (CBCT) images. MATERIALS AND METHODS: Facial asymmetry was analysed in a total of 100 Class III patients aged 16 years or older using CBCT scans. Patients were categorized into subgroups based on asymmetry pattern. Measurements of the nasomaxillary complex were obtained from the CBCT scans, including the volume and width of the maxillary sinuses and nasal cavities on deviated and non-deviated sides, as well as the displacement of the nasal septum. Statistical analysis was performed to compare the internal nasomaxillary variables within and between groups, and regression analysis was conducted to evaluate the correlation between facial asymmetry and the internal nasomaxillary variables. RESULTS: Group comparisons showed that there were no significant differences in the volume of the maxillary sinus and nasal cavity. However, the direction and extent of nasal septum deviation, as well as the width of the nasal cavity, varied depending on the maxillary asymmetry pattern. Regression analysis indicated a correlation between nasal septum deviation and the difference in maxillary height, while the difference in nasal cavity width was correlated with the difference in maxillary width. CONCLUSION: A comprehensive evaluation of the internal nasal anatomy is vital for understanding the intricate relationship between nasal structure and maxillary growth.

Significant Reduction in External Ventricular Drain-Related Infections After Introducing a Novel Bundle Protocol: A Before and After Trial.

BACKGROUND: External ventricular drain (EVD)-related infection (ERI) is a serious complication in neurosurgical patients. The estimated ERI rates range from 5 to 20 cases per 1,000 EVD catheter days. The pathophysiology of ERI is similar to central line-associated bloodstream infections (CLABSIs) stemming from skin-derived bacterial colonization. The use of bundle management can reduce CLABSI rates. Due to the pathogenic similarities between infections related to the two devices, we developed and evaluated the effectiveness of an ERI-bundle protocol based on CLABSI bundles. METHODS: From November 2016 to November 2021, we conducted a study to evaluate the effectiveness of an ERI-bundle protocol. This study adopted a before-and-after trial, comparing the ERI rates for the 2 years before and 3 years after the introduction of the newly developed ERI-bundle protocol. We also analyzed the contributing factors to ERI using logistic regression analysis. RESULTS: A total of 183 patients with 2,381 days of catheter use were analyzed. The ERI rate decreased significantly after the ERI-bundle protocol from 16.7% (14 of 84; 14.35 per 1,000 catheter days) to 4.0% (4 of 99; 3.21 per 1,000 catheter days) (P = 0.004). CONCLUSION: Introduction of the ERI-bundle protocol was very effective in reducing ERI.

Firing of Replication Origins Is Disturbed by a CDK4/6 Inhibitor in a pRb-Independent Manner.

Over the last decade, CDK4/6 inhibitors (palbociclib, ribociclib and abemaciclib) have emerged as promising anticancer drugs. Numerous studies have demonstrated that CDK4/6 inhibitors efficiently block the pRb-E2F pathway and induce cell cycle arrest in pRb-proficient cells. Based on these studies, the inhibitors have been approved by the FDA for treatment of advanced hormonal receptor (HR) positive breast cancers in combination with hormonal therapy. However, some evidence has recently shown unexpected effects of the inhibitors, underlining a need to characterize the effects of CDK4/6 inhibitors beyond pRb. Our study demonstrates how palbociclib impairs origin firing in the DNA replication process in pRb-deficient cell lines. Strikingly, despite the absence of pRb, cells treated with palbociclib synthesize less DNA while showing no cell cycle arrest. Furthermore, this CDK4/6 inhibitor treatment disturbs the temporal program of DNA replication and reduces the density of replication forks. Cells treated with palbociclib show a defect in the loading of the Pre-initiation complex (Pre-IC) proteins on chromatin, indicating a reduced initiation of DNA replication. Our findings highlight hidden effects of palbociclib on the dynamics of DNA replication and of its cytotoxic consequences on cell viability in the absence of pRb. This study provides a potential therapeutic application of palbociclib in combination with other drugs to target genomic instability in pRB-deficient cancers.

Correlation between craniofacial changes and respiratory improvement after nasomaxillary skeletal expansion in pediatric obstructive sleep apnea patients.

PURPOSE: The aim of this study was to investigate the correlation between the changes in respiratory function and dimensions of the nasomaxillary complex (NMC) and upper airway (UA) compartments after nasomaxillary skeletal expansion (NMSE) treatment for pediatric patients with obstructive sleep apnea (OSA). METHODS: Nonobese OSA patients (mean age, 13.6 ± 2.9 years; mean body mass index, 18.1 ± 3.0 kg/m2); mean apnea-hypopnea index (AHI, 7.0 ± 5.4 events/h) presenting with transverse nasomaxillary constriction were evaluated before and after NMSE using cone-beam computed tomography (CBCT), home sleep test, and modified pediatric sleep questionnaire (m-PSQ). Paired t tests were performed to examine the treatment-related changes in all the parameters, and a multiple regression analysis adjusted for age and sagittal and vertical skeletal patterns was conducted to determine the dimensional parameters to affect the functional improvement. RESULTS: Among 26 patients, NMSE treatment significantly increased NMC dimensions at all tested levels and all UA compartments in CBCT, except glossopharyngeal airway. Concurrently, AHI, oxygen desaturation index, the lowest oxygen saturation (LSaO2), flow limitation (FL), snoring, and m-PSQ were significantly improved. AHI reduction was correlated with UA enlargement with no correlation with NMC expansion, whereas FL reduction was affected by NMC expansion. The minimal cross-sectional area was the most predictive of functional improvement, presenting correlations with AHI, LSaO2, and m-PSQ. CONCLUSION: NMSE can be a good treatment for pediatric OSA patients when applied to enhance the nasal and pharyngeal airway patencies beyond the NMC, ultimately to improve pharyngeal collapsibility as well as nasal airflow.

Genetic effect of single nucleotide polymorphisms in growth hormone receptor gene on the risk of non-syndromic mandibular prognathism in the Korean population.

OBJECTIVES: To evaluate the association of three single-nucleotide polymorphisms (SNPs) of growth hormone receptor (GHR) gene with mandibular prognathism (MP) and relationships between mandibular morphology and GHR gene SNPs in the Korean population. MATERIALS AND METHODS: A total of 325 subjects were divided into two groups based on sagittal maxillomandibular relationship by the lateral cephalography: the MP and control groups. From the SNPs in the GHR gene, three SNPs (rs6180, rs6182 and rs6184) were selected. SNP genotyping was performed using direct sequencing. The craniofacial measurements of lateral cephalography were analysed. RESULTS: We found a lack of association between GHR and MP. However, in the analysis according to the values of cephalometric measurements, rs6180 was significantly associated with ANB, SNB, effective mandibular length and SNMP in females. Additionally, rs6182 and rs6184 were significantly associated with ramal height in males. CONCLUSION: Growth hormone receptor SNPs may affect not only the sagittal development of mandible but also the vertical development of ramal height, and GHR SNPs may gender-differently influence mandibular morphology. This finding supports that the GHR might be susceptible on mandibular morphogenesis in the Korean population.

Effect of locally delivered protein complex-loaded nanoparticles on bone remodelling of atrophic alveolar ridge in beagles.

OBJECTIVE: To investigate the effect of local injection of mineralized hybrid nanoparticles loading dentin matrix protein-1 (DMP-1) and matrix metalloproteinase-13 (MMP-13) complex (P-NPs) on the bone remodelling on atrophic alveolar ridges (AAR) ahead of orthodontic tooth movement (OTM). SETTINGS AND SAMPLE POPULATION: Four beagles were randomly allocated into Group C (OTM only) and Group NP (OTM with P-NPs injection). Experimental model of AAR was prepared in 8 mandibular quadrants after extraction of the third premolars (n = 4 per Group). MATERIALS AND METHODS: Reciprocal traction of the second and fourth premolars was performed towards AAR for 8 weeks. P-NPs were prepared by loading recombinant DMP-1 and MMP-13 complex into calcium carbonate (CaCO3 )-mineralized hybrid nanoparticles and injected at 0, 3 and 6 weeks. The rate of OTM and the bone remodelling characteristics were compared between Groups using fluorescent microscopic analysis and microstructural histomorphometric analysis. RESULTS: Group NP revealed higher bone volume fraction and higher trabecular ratio with lower bone mineral density than Group C on AAR area. Meanwhile, the root movement towards AAR was facilitated in Group NP representing more bodily movement than Group C. CONCLUSION: Non-invasive intervention of P-NPs injection suggested a clinical potential to facilitate translational movement into the AAR with sustaining woven bone-like microstructural environment.

Tumor Promoting Effects of Sulforaphane on Diethylnitrosamine-Induced Murine Hepatocarcinogenesis.

Nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor involved in protection against initiation of carcinogenesis in normal cells. Notably, recent studies have demonstrated that aberrant activation of NRF2 accelerates the proliferation and progression of cancer cells. The differential effects of NRF2 on multi-stage carcinogenesis have raised a concern about the validity of NRF2 activators for chemoprevention. This prompted us to assess the effects of sulforaphane (SFN), a prototypic NRF2 activating chemopreventive phytochemical, on experimentally induced carcinogenesis. In the present study, SFN was daily injected intraperitoneally (25 mg/kg) for 3 months to male C57BL/6 mice at 6 months after single intraperitoneal administration of a hepatocarcinogen, diethylnitrosamine (DEN). The liver to body weight ratio, tumor growth, and the number and the size of hepatomas measured at 9 months after DEN administration were significantly higher in SFN-treated mice than those in vehicle-treated mice. Moreover, the expression of NRF2, its target protein NAD(P)H:quinone oxidoreductase 1, and the cell proliferation marker, proliferating cell nuclear antigen was further elevated in DEN plus SFN-treated mice. These results suggest that once hepatocarcinogenesis is initiated, SFN may stimulate tumor progression.

Accuracy of convolutional neural networks-based automatic segmentation of pharyngeal airway sections according to craniofacial skeletal pattern.

INTRODUCTION: This study aimed to evaluate a 3-dimensional (3D) U-Net-based convolutional neural networks model for the fully automatic segmentation of regional pharyngeal volume of interests (VOIs) in cone-beam computed tomography scans to compare the accuracy of the model performance across different skeletal patterns presenting with various pharyngeal dimensions. METHODS: Two-hundred sixteen cone-beam computed tomography scans of adult patients were randomly divided into training (n = 100), validation (n = 16), and test (n = 100) datasets. We trained the 3D U-Net model for fully automatic segmentation of pharyngeal VOIs and their measurements: nasopharyngeal, velopharyngeal, glossopharyngeal, and hypopharyngeal sections as well as total pharyngeal airway space (PAS). The test datasets were subdivided according to the sagittal and vertical skeletal patterns. The segmentation performance was assessed by dice similarity coefficient, volumetric similarity, precision, and recall values, compared with the ground truth created by 1 expert's manual processing using semiautomatic software. RESULTS: The proposed model achieved highly accurate performance, showing a mean dice similarity coefficient of 0.928 ± 0.023, the volumetric similarity of 0.928 ± 0.023, precision of 0.925 ± 0.030, and recall of 0.921 ± 0.029 for total PAS segmentation. The performance showed region-specific differences, revealing lower accuracy in the glossopharyngeal and hypopharyngeal sections than in the upper sections (P <0.001). However, the accuracy of model performance at each pharyngeal VOI showed no significant difference according to sagittal or vertical skeletal patterns. CONCLUSIONS: The 3D-convolutional neural network performance for region-specific PAS analysis is promising to substitute for laborious and time-consuming manual analysis in every skeletal and pharyngeal pattern.

Craniofacial treatment protocol for a pediatric patient with obstructive sleep apnea and skeletal Class III malocclusion: A 13-year follow-up.

This report aimed to describe the long-term effects of craniofacial growth modification treatment on sleep and breathing functions in a 7-year-old girl diagnosed with skeletal Class III malocclusion and sleep-disordered breathing. Based on the flowchart of orthodontic intervention protocol that we proposed for phenotype-based patient selection and skeletal target-based treatment selection for pediatric patients with sleep-disordered breathing, a 2-phase treatment targeting the nasomaxillary complex was performed. Posttreatment 3-dimensional changes in the skeletal structure and upper airway were evaluated in association with functional assessment using a validated pediatric sleep questionnaire and home sleep test. Esthetic improvement and obstructive sleep apnea cure were achieved without skeletal surgery. The 2-year retention records showed stable occlusion and improved facial profile with normal breathing and sleep.

Accuracy of auto-identification of the posteroanterior cephalometric landmarks using cascade convolution neural network algorithm and cephalometric images of different quality from nationwide multiple centers.

INTRODUCTION: The purpose of this study was to evaluate the accuracy of auto-identification of the posteroanterior (PA) cephalometric landmarks using the cascade convolution neural network (CNN) algorithm and PA cephalogram images of a different quality from nationwide multiple centers nationwide. METHODS: Of the 2798 PA cephalograms from 9 university hospitals, 2418 images (2075 training set and 343 validation set) were used to train the CNN algorithm for auto-identification of 16 PA cephalometric landmarks. Subsequently, 99 pretreatment images from the remaining 380 test set images were used to evaluate the accuracy of auto-identification of the CNN algorithm by comparing with the identification by a human examiner (gold standard) using V-Ceph 8.0 (Ostem, Seoul, South Korea). Pretreatment images were used to eliminate the effects of orthodontic bracket, tube and wire, surgical plate, and surgical screws. Paired t test was performed to compare the x- and y-coordinates of each landmark. The point-to-point error and the successful detection rate (range, within 2.0 mm) were calculated. RESULTS: The number of landmarks without a significant difference between the location identified by the human examiner and by auto-identification by the CNN algorithm were 8 on the x-coordinate and 5 on the y-coordinate, respectively. The mean point-to-point error was 1.52 mm. The low point-to-point error (<1.0 mm) was observed at the left and right antegonion (0.96 mm and 0.99 mm, respectively) and the high point-to-point error (>2.0 mm) was observed at the maxillary right first molar root apex (2.18 mm). The mean successful detection rate of auto-identification was 83.3%. CONCLUSIONS: Cascade CNN algorithm for auto-identification of PA cephalometric landmarks showed a possibility of an effective alternative to manual identification.

Vertical bony step between proximal and distal segments after mandibular setback is related with relapse: A cone-beam computed tomographic study.

INTRODUCTION: Vertical bony step (VBS) occurs between proximal and distal segments of the mandible during mandibular setback surgery with bilateral sagittal split ramus osteotomy. The purpose of this study was to investigate whether VBS is correlated with the relapse of mandibular setback using 3-dimensional models constructed from cone-beam computed tomography. METHODS: The subjects consisted of 30 patients who underwent bilateral sagittal split ramus osteotomy for a mandibular setback. Double jaw surgery was performed in 18 patients, and isolated mandibular setback surgery was performed in 12 patients. Cone-beam computed tomography scans were taken at pretreatment (T0), postsurgery (T1), and posttreatment (T2). Treatment changes and the correlations between measurements were evaluated. RESULTS: The mean mandibular setback was -11.9 mm, and the mean VBS was -5.6 mm. Correlations with the relapse of mandibular setback were found in the amount of mandibular setback (T1 - T0), development of VBS (T1 - T0), posterior movement of the proximal segment (T1 - T0), counterclockwise rotation of symphysis (T2 - T1), and the resolution of VBS (T2 - T1). CONCLUSIONS: The development and resolution of VBS were correlated with the relapse of mandibular setback. Minimizing VBS is recommended to reduce the relapse of mandibular setback.

Fully automated identification of cephalometric landmarks for upper airway assessment using cascaded convolutional neural networks.

OBJECTIVES: The aim of the study was to evaluate the accuracy of a cascaded two-stage convolutional neural network (CNN) model in detecting upper airway (UA) soft tissue landmarks in comparison with the skeletal landmarks on the lateral cephalometric images. MATERIALS AND METHODS: The dataset contained 600 lateral cephalograms of adult orthodontic patients, and the ground-truth positions of 16 landmarks (7 skeletal and 9 UA landmarks) were obtained from 500 learning dataset. We trained a UNet with EfficientNetB0 model through the region of interest-centred circular segmentation labelling process. Mean distance errors (MDEs, mm) of the CNN algorithm was compared with those from human examiners. Successful detection rates (SDRs, per cent) assessed within 1-4 mm precision ranges were compared between skeletal and UA landmarks. RESULTS: The proposed model achieved MDEs of 0.80 ± 0.55 mm for skeletal landmarks and 1.78 ± 1.21 mm for UA landmarks. The mean SDRs for UA landmarks were 72.22 per cent for 2 mm range, and 92.78 per cent for 4 mm range, contrasted with those for skeletal landmarks amounting to 93.43 and 98.71 per cent, respectively. As compared with mean interexaminer difference, however, this model showed higher detection accuracies for geometrically constructed UA landmarks on the nasopharynx (AD2 and Ss), while lower accuracies for anatomically located UA landmarks on the tongue (Td) and soft palate (Sb and St). CONCLUSION: The proposed CNN model suggests the availability of an automated cephalometric UA assessment to be integrated with dentoskeletal and facial analysis.

Sphingomyelin synthase 1 mediates hepatocyte pyroptosis to trigger non-alcoholic steatohepatitis.

OBJECTIVE: Lipotoxic hepatocyte injury is a primary event in non-alcoholic steatohepatitis (NASH), but the mechanisms of lipotoxicity are not fully defined. Sphingolipids and free cholesterol (FC) mediate hepatocyte injury, but their link in NASH has not been explored. We examined the role of free cholesterol and sphingomyelin synthases (SMSs) that generate sphingomyelin (SM) and diacylglycerol (DAG) in hepatocyte pyroptosis, a specific form of programmed cell death associated with inflammasome activation, and NASH. DESIGN: Wild-type C57BL/6J mice were fed a high fat and high cholesterol diet (HFHCD) to induce NASH. Hepatic SMS1 and SMS2 expressions were examined in various mouse models including HFHCD-fed mice and patients with NASH. Pyroptosis was estimated by the generation of the gasdermin-D N-terminal fragment. NASH susceptibility and pyroptosis were examined following knockdown of SMS1, protein kinase Cδ (PKCδ), or the NLR family CARD domain-containing protein 4 (NLRC4). RESULTS: HFHCD increased the hepatic levels of SM and DAG while decreasing the level of phosphatidylcholine. Hepatic expression of Sms1 but not Sms2 was higher in mouse models and patients with NASH. FC in hepatocytes induced Sms1 expression, and Sms1 knockdown prevented HFHCD-induced NASH. DAG produced by SMS1 activated PKCδ and NLRC4 inflammasome to induce hepatocyte pyroptosis. Depletion of Nlrc4 prevented hepatocyte pyroptosis and the development of NASH. Conditioned media from pyroptotic hepatocytes activated the NOD-like receptor family pyrin domain containing 3 inflammasome (NLRP3) in Kupffer cells, but Nlrp3 knockout mice were not protected against HFHCD-induced hepatocyte pyroptosis. CONCLUSION: SMS1 mediates hepatocyte pyroptosis through a novel DAG-PKCδ-NLRC4 axis and holds promise as a therapeutic target for NASH.

Naphthalimide-4-(4-nitrophenyl)thiosemicarbazide: A Fluorescent Probe for Simultaneous Monitoring of Viscosity and Nitric Oxide in Living Cells.

Intracellular viscosity is a physicochemical factor that determines the outcomes of various biological processes, while nitric oxide (NO) is an essential signaling molecule that controls many cellular processes, including oxidative stress. Anticipating that both may be interrelated with a variety of pathologies, their simultaneous measurement would be highly valuable for the investigation of the pathological condition of cells. However, the development of a sensor for such simultaneous detection has not been attempted yet. Herein, we present the synthesis of naphthalimide-4-(4-nitrophenyl)thiosemicarbazide, probe 1, and its application to living cells under conditions of lipopolysaccharide or nystatin treatment, adopted as oxidative stress and altered intracellular viscosity models, respectively. The probe showed increased fluorescence in response to elevation of viscosity and NO levels at 470 and 550 nm, respectively, in the solution studies. When the probe was used for a confocal microscopic study of HeLa cells under stressed conditions, simultaneous monitoring of viscosity and NO level elevations was possible through fluorescence imaging using band-pass filters of 420-475 and 505-600 nm, respectively, upon excitation at a wavelength of 405 nm. Interestingly, both the cellular viscosity and NO levels increased together under lipopolysaccharide or nystatin treatment. Therefore, we suggest that probe 1 is a fluorescent chemical probe that enables the monitoring of alterations in intracellular viscosity and NO levels in living cells, which would be valuable in studies of various cellular damage models.

Alternative regulation of HIF-1α stability through Phosphorylation on Ser451.

The hypoxia-inducible factor (HIF-1α) functions as a master regulator of oxygen homeostasis. Oxygen-dependent hydroxylation of HIF-1α is tightly regulated by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). The prolyl hydroxylation facilitates the recruitment of the von Hippel-Lindau (VHL) protein, leading to ubiquitination and degradation of HIF-1α by the proteasomes. Besides prolyl hydroxylation, phosphorylation of HIF-1α is another central post-translational modification, which regulates its stability under hypoxic conditions as well as normoxic conditions. By use of LC/MS/MS-based analysis, we were able to identify a specific serine residue (Ser451) of HIF-1α phosphorylated under hypoxic conditions. Using plasmids expressing wild type (WT), non-phosphorylatable mutant HIF-1α (S451A), and phosphomimetic mutant HIF-1α (S451E), we demonstrated that the phosphorylation at Ser451 is important in maintaining the HIF-1α protein stability. Notably, phosphorylation at S451 interrupts the interaction of HIF-1α with PHD and pVHL. A phosphomimetic construct of HIF-1α at Ser451 (S451E) is significantly more stable than WT HIF-1α under normoxic conditions. Cells transfected with unphosphorylatable HIF-1α exhibited significantly lower HIF-1 transcriptional activity than WT cells and markedly reduced tumor cell migration. Further, tumors derived from the phosphomimetic mutant cells grew faster, whereas the tumors derived from non-phosphorylatable mutant cells grew slower than the control tumors, suggesting that the phosphorylation of HIF-1α at the Ser451 site is critical to promote tumor growth in vivo. Taken together, our data suggest an alternative mechanism responsible for the regulation of HIF-1α stability.

Stabilization of C/EBPß through direct interaction with STAT3 in H-Ras transformed human mammary epithelial cells.

Signal transducer and activator of transcription 3 (STAT3) plays important roles in cancer-associated inflammation by controlling expression of proinflammatory cytokines and chemokines. Recent studies suggest that C/EBPß (CCAAT-enhancer binding protein beta) and STAT3 synergistically stimulate cancer cell proliferation and epithelial-mesenchymal transition. C/EBPß is a leucine-zipper transcription factor that regulates expression of a variety of inflammatory cytokines or chemokines, such as IL-8, G-CSF (granulocyte colony stimulating factor), and GM-CSF (granulocyte macrophage colony stimulating factor) which induce neutrophil infiltration and differentiation. However, molecular mechanisms by which STAT3 and C/EBPß cooperatively interact had not been fully elucidated. In this study, we found that the level of C/EBPß protein, but not that of its mRNA transcript, was decreased in the absence of STAT3 in H-Ras transformed human mammary epithelial (H-Ras MCF10A) cells. In addition, silencing STAT3 dramatically induced ubiquitination of C/EBPß for proteasomal degradation. Furthermore, direct interaction between STAT3 and C/EBPß was confirmed by immunoprecipitation and proximity ligation assays. Taken together, these results suggest that STAT3 stabilizes C/EBPß, thereby promoting cancer-associated inflammation.