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Abstract Cancers with activating mutations of KRAS show a high prevalence but remain intractable, requiring innovative strategies to overcome the poor targetability of KRAS. Here, we report that KRAS expression is post-translationally up-regulated through deubiquitination when the scaffolding function of NDRG3 (N-Myc downstream-regulated gene 3) promotes specific interaction between KRAS and a deubiquitinating enzyme, USP9X. In KRAS-mutant cancer cells KRAS protein expression, downstream signaling, and cell growth are highly dependent on NDRG3. In conditional KrasG12D knock-in mouse models of pancreatic ductal adenocarcinoma, Ndrg3 depletion abolishes Kras protein expression and suppresses intraepithelial neoplasia formation in pancreas. Mechanistically, KRAS protein binds to the C-terminal serine/threonine-rich region of NDRG3, subsequently going through deubiquitination by USP9X recruited to the complex. This interaction can be disrupted in a dominant-negative manner by a C-terminal NDRG3 fragment that binds KRAS but is defective in USP9X binding, highly suppressing KRAS protein expression and KRAS-driven cell growth. In summary, KRAS-driven cancer development critically depends on the deubiquitination of KRAS protein mediated by USP9X/NDRG3, and KRAS-addicted cancers could be effectively targeted by inhibiting the KRAS-NDRG3 interaction.

Background/Objectives: Occlusion plays a crucial role in the long-term success and functionality of dental restorations. The purpose of this study was to investigate the changes in occlusal contacts upon the cementation of zirconia crowns with different cement spacer settings in computer-aided design and computer-aided manufacturing (CAD-CAM) software (3Shape Dental System version 2.102.1.0). Methods: A master model of a prepared abutment for a crown on the right maxillary first molar was scanned, and 30 sets of sample casts and zirconia crowns were fabricated with varying cement spaces (70 μm and 120 μm). These casts were mounted in maximal intercuspation (MIP) on a semi-adjustable articulator. Pre-cementation adjustments were made to fit the crowns and maintain the existing occlusion. Occlusal records were taken before and after cementation using polyvinyl siloxane impression material. These records were analyzed using a DC light box and image analyzer to measure changes in contact area, intensity, and patterns. Paired sample t-tests were used to compare pre- and post-cementation occlusal contact areas of each sample (α = 0.05). Results: Significant differences in occlusal contact areas were found between pre- and post-cementation in both groups (p < 0.001). The mean post-cementation contact surface area for the 70 μm group was 6281 ± 3310 μm2, compared to 2339 ± 1206 μm2 before cementation. For the 120 μm group, the post-cementation area was 5545 ± 3491 μm2, compared to 2071 ± 909 μm2 before cementation. An increase in contact intensity was also observed after cementation. Conclusions: This study demonstrates that cementation increases occlusal contact surface area and intensity in both cement space groups.

Traditionally, metal-ceramics, metal-reinforced acrylics, and--more recently--full-contour or layered zirconia have been the materials of choice for definitive fixed implant-supported rehabilitations. Polymethyl methacrylate (PMMA) is commonly used in implant dentistry for the fabrication of implant-supported interim prostheses and as milled or 3D-printed prototypes. This article describes a novel protocol to prosthetically restore a completely edentulous patient following a digital workflow, with fixed, screw-retained, implant-supported prostheses fabricated from CAD/CAM milled PMMA, with no metal substructure. After a 2-year follow-up in terms of esthetics, phonetics, function, and biologic tissue response, the outcome remains functional and free of mechanical, biomechanical, or biologic complications. The aim of this article is to illustrate the feasibility of using milled PMMA as a viable definitive prosthetic material for the fixed implant rehabilitation of edentulous patients. [ABSTRACT FROM AUTHOR]

Cancers with activating mutations of KRAS show a high prevalence but remain intractable, requiring innovative strategies to overcome the poor targetability of KRAS. Here, we report that KRAS expression is post-translationally up-regulated through deubiquitination when the scaffolding function of NDRG3 (N-Myc downstream-regulated gene 3) promotes specific interaction between KRAS and a deubiquitinating enzyme, USP9X. In KRAS-mutant cancer cells KRAS protein expression, downstream signaling, and cell growth are highly dependent on NDRG3. In conditional KrasG12D knock-in mouse models of pancreatic ductal adenocarcinoma, Ndrg3 depletion abolishes Kras protein expression and suppresses intraepithelial neoplasia formation in pancreas. Mechanistically, KRAS protein binds to the C-terminal serine/threonine-rich region of NDRG3, subsequently going through deubiquitination by USP9X recruited to the complex. This interaction can be disrupted in a dominant-negative manner by a C-terminal NDRG3 fragment that binds KRAS but is defective in USP9X binding, highly suppressing KRAS protein expression and KRAS-driven cell growth. In summary, KRAS-driven cancer development critically depends on the deubiquitination of KRAS protein mediated by USP9X/NDRG3, and KRAS-addicted cancers could be effectively targeted by inhibiting the KRAS-NDRG3 interaction. The regulation of KRAS oncoprotein stability remains to be completely determined. Here the authors identify that USP9X-mediated deubiquitination of KRAS protein is dependent on scaffolding protein NDRG3, suggesting KRAS-NDRG3 interaction as a targetable vulnerability in KRAS-driven cancers. [ABSTRACT FROM AUTHOR]

Recent advances in cryo-electron microscopy (cryo-EM) have facilitated the high-resolution structural determination of macromolecular complexes in their native states, providing valuable insights into their dynamic behaviors. However, insufficient understanding or experience with the cryo-EM image processing parameters can result in the loss of biological meaning. In this paper, we investigate the dihydrolipoyl acetyltransferase (E2) inner core complex of the pyruvate dehydrogenase complex (PDC) and reconstruct the 3D maps using five different symmetry parameters. The results demonstrate that the reconstructions yield structurally identical 3D models even at a near-atomic structure. This finding underscores a crucial message for researchers engaging in single-particle analysis (SPA) with relatively user-friendly and convenient image processing software. This approach helps reduce the risk of missing critical biological details, such as the dynamic properties of macromolecules. [ABSTRACT FROM AUTHOR]

Background/Objectives: Occlusion plays a crucial role in the long-term success and functionality of dental restorations. The purpose of this study was to investigate the changes in occlusal contacts upon the cementation of zirconia crowns with different cement spacer settings in computer-aided design and computer-aided manufacturing (CAD-CAM) software (3Shape Dental System version 2.102.1.0). Methods: A master model of a prepared abutment for a crown on the right maxillary first molar was scanned, and 30 sets of sample casts and zirconia crowns were fabricated with varying cement spaces (70 μm and 120 μm). These casts were mounted in maximal intercuspation (MIP) on a semi-adjustable articulator. Pre-cementation adjustments were made to fit the crowns and maintain the existing occlusion. Occlusal records were taken before and after cementation using polyvinyl siloxane impression material. These records were analyzed using a DC light box and image analyzer to measure changes in contact area, intensity, and patterns. Paired sample t-tests were used to compare pre- and post-cementation occlusal contact areas of each sample (α = 0.05). Results: Significant differences in occlusal contact areas were found between pre- and post-cementation in both groups (p < 0.001). The mean post-cementation contact surface area for the 70 μm group was 6281 ± 3310 μm2, compared to 2339 ± 1206 μm2 before cementation. For the 120 μm group, the post-cementation area was 5545 ± 3491 μm2, compared to 2071 ± 909 μm2 before cementation. An increase in contact intensity was also observed after cementation. Conclusions: This study demonstrates that cementation increases occlusal contact surface area and intensity in both cement space groups. [ABSTRACT FROM AUTHOR]

Background/Aims Coenzyme Q10 (CoQ10), is a promising antioxidant; however, low bioavailability owing to lipid-solubility is a limiting factor. We developed water-soluble CoQ10 (CoQ10-W) and compared its effects with conventional lipid-soluble CoQ10 (CoQ10-L) in an experimental model of chronic tacrolimus (Tac) nephropathy. Methods CoQ10-W was developed from a glycyrrhizic-carnitine mixed layer CoQ10 micelle based on acyltransferases. Chronic nephropathy was induced in rats with 28-day Tac treatment; they were concomitantly treated with CoQ10-L or CoQ10-W. CoQ10 level in plasma and kidney were measured using liquid chromatography–mass spectrometry. CoQ10-W and CoQ10-L effects on Tac-induced nephropathy were assessed in terms of renal function, histopathology, oxidative stress, and apoptotic cell death. Their effects on cell viability and reactive oxygen species (ROS) production were assessed in cultured proximal tubular cells, human kidney 2 (HK-2) cells. Results The plasma CoQ10 level was significantly higher in the CoQ10-W group than in the CoQ10-L group. Tac treatment caused renal dysfunction, typical pathologic lesions, and oxidative stress markers. Serum creatinine was restored in the Tac + CoQ10-L or CoQ10-W groups compared with that in the Tac group. CoQ10-W administration reduced oxidative stress and apoptosis markers. Mitochondrial ultrastructure assessment revealed that the addition of CoQ10-L or CoQ10-W with Tac increased mitochondrial size and number than Tac treatment alone. In vitro investigations revealed that both CoQ10-L and CoQ10-W improved cell viability and reduced ROS production in the Tac-induced HK-2 cell injury. Conclusions CoQ10-W has a better therapeutic effect in Tac-induced renal injury than conventional CoQ10-L, possibly associated with improved CoQ10 bioavailability

Background/Aims Coenzyme Q10 (CoQ10) has antioxidant effects and is commercially available and marketed extensively. However, due to its low bioavailability, its effects are still controversial. We developed a water-soluble CoQ10-based micelle formulation (CoQ10-W) and tested it in an experimental model of tacrolimus (TAC)-induced diabetes mellitus (DM). Methods We developed CoQ10-W from a glycyrrhizic-carnitine mixed layer CoQ10 micelle preparation based on acyltransferases. TAC-induced DM rats were treated with either lipid-soluble CoQ10 (CoQ10-L) or CoQ10-W for 4 weeks. Their plasma and pancreatic CoQ10 concentrations were measured using liquid chromatography- tandem mass spectrometry. The therapeutic efficacies of CoQ10-W and CoQ10-L on TAC-induced DM were compared using functional and morphological parameters and their effects on cell viability and reactive oxygen species (ROS) production were also evaluated in cultured rat insulinoma cells. Results The plasma CoQ10 level was significantly increased in the CoQ10-W group compared to that in the CoQ10-L group. Intraperitoneal glucose tolerance tests and glucose-stimulated insulin secretion revealed that CoQ10-W controlled hyperglycemia and restored insulin secretion significantly better than CoQ10-L. The TAC-mediated decrease in pancreatic islet size was significantly attenuated by CoQ10-W but not by CoQ10-L. TAC-induced oxidative stress and apoptosis were significantly more reduced by CoQ10-W than CoQ10-L. Electron microscopy revealed that CoQ10-W restored TAC-induced attenuation in the number of insulin granules and the average mitochondrial area, unlike CoQ10-L. In vitro studies showed that CoQ10-L and CoQ10-W both improved cell viability and reduced ROS production in TAC-treated islet cells to a similar extent. Conclusions CoQ10-W has better therapeutic efficacy than CoQ10-L in TAC-induced DM.

Abstract Background Fibrosis is an integral component of the pathogenesis of acute lung injury and is associated with poor outcomes in patients with acute respiratory distress syndrome (ARDS). Fibrocytes are bone marrow-derived cells that traffic to injured tissues and contribute to fibrosis; hence their concentration in the peripheral blood has the potential to serve as a biomarker of lung fibrogenesis. We therefore sought to test the hypothesis that the concentration and phenotype of circulating fibrocytes in patients with ARDS predicts clinical outcomes. Methods For the animal studies, C57Bl/6 mice were infected with experimental Klebsiella pneumoniae in a model of acute lung injury; one-way ANOVA was used to compare multiple groups and two-way ANOVA was used to compare two groups over time. For the human study, 42 subjects with ARDS and 12 subjects with pneumonia (without ARDS) were compared to healthy controls. Chi-squared or Fisher’s exact test were used to compare binary outcomes. Survival data was expressed using a Kaplan-Meier curve and compared by log-rank test. Univariable and multivariable logistic regression were used to predict death. Results In mice with acute lung injury caused by Klebsiella pneumonia, there was a time-dependent increase in lung soluble collagen that correlated with sequential expansion of fibrocytes in the bone marrow, blood, and then lung compartments. Correspondingly, when compared via cross-sectional analysis, the initial concentration of blood fibrocytes was elevated in human subjects with ARDS or pneumonia as compared to healthy controls. In addition, fibrocytes from subjects with ARDS displayed an activated phenotype and on serial measurements, exhibited intermittent episodes of markedly elevated concentration over a median of 1 week. A peak concentration of circulating fibrocytes above a threshold of > 4.8 × 106 cells/mL cells correlated with mortality that was independent of age, ratio of arterial oxygen concentration to the fraction of inspired oxygen, and vasopressor requirement. Conclusions Circulating fibrocytes increase in a murine model of acute lung injury and elevation in the number of these cells above a certain threshold is correlated with mortality in human ARDS. Therefore, these cells may provide a useful and easily measured biomarker to predict outcomes in these patients.

BackgroundOsteoarthritis (OA) is the most common degenerative joint disease and is characterized by breakdown of joint cartilage. Coenzyme Q10 (CoQ10) exerts diverse biological effects on bone and cartilage; observational studies have suggested that CoQ10 may slow OA progression and inflammation. However, any effect of CoQ10 on OA remains unclear. Here, we investigated the therapeutic utility of CoQ10-micelles.MethodsSeven-week-old male Wistar rats were injected with monosodium iodoacetate (MIA) to induce OA. CoQ10-micelles were administered orally to MIA-induced OA rats; celecoxib served as the positive control. Pain, tissue destruction, and inflammation were measured. The expression levels of catabolic and inflammatory cell death markers were assayed in CoQ10-micelle-treated chondrocytes.ResultsOral supplementation with CoQ10-micelles attenuated OA symptoms remarkably, including pain, tissue destruction, and inflammation. The expression levels of the inflammatory cytokines IL-1β, IL-6, and MMP-13, and of the inflammatory cell death markers RIP1, RIP3, and pMLKL in synovial tissues were significantly reduced by CoQ10-micelle supplementation, suggesting that CoQ10-micelles might attenuate the synovitis of OA. CoQ10-micelle addition to cultured OA chondrocytes reduced the expression levels of catabolic and inflammatory cell death markers.ConclusionsCoQ10-micelles might usefully treat OA.