Oncologic Outcomes in Nipple-sparing Mastectomy with Immediate Reconstruction and Total Mastectomy with Immediate Reconstruction in Women with Breast Cancer: A Machine-Learning Analysis.

BACKGROUND: This study used a single-institution cohort, the Severance dataset, validated the results by using the surveillance, epidemiology, and end results (SEER) database, adjusted with propensity-score matching (PSM), and analyzed by using a machine learning method. To determine whether the 5-year, disease-free survival (DFS) and overall survival (OS) of patients undergoing nipple-sparing mastectomy (NSM) with immediate breast reconstruction (IBR) are not inferior to those of women treated with total mastectomy/skin-sparing mastectomy (TM/SSM). METHODS: The Severance dataset enrolled 611 patients with early, invasive breast cancer from 2010 to 2017. The SEER dataset contained data for 485,245 patients undergoing TM and 14,770 patients undergoing NSM between 2000 and 2018. All patients underwent mastectomy and IBR. Intraoperative, frozen-section biopsy for the retro-areolar tissue was performed in the NSM group. The SEER dataset was extracted by using operation types, including TM/SSM and NSM. The primary outcome was DFS for the Severance dataset and OS for the SEER dataset. PSM analysis was applied. Survival outcomes were analyzed by using the Kaplan-Meier method and Cox proportional hazard (Cox PH) regression model. We implemented XGBSE to predict mortality with high accuracy and evaluated model prediction performance using a concordance index. The final model inspected the impact of relevant predictors on the model output using shapley additive explanation (SHAP) values. RESULTS: In the Severance dataset, 151 patients underwent NSM with IBR and 460 patients underwent TM/SSM with IBR. No significant differences were found between the groups. In multivariate analysis, NSM was not associated with reduced oncologic outcomes. The same results were observed in PSM analysis. In the SEER dataset, according to the SHAP values, the individual feature contribution suggested that AJCC stage ranks first. Analyses from the two datasets confirmed no impact on survival outcomes from the two surgical methods. CONCLUSIONS: NSM with IBR is a safe and feasible procedure in terms of oncologic outcomes. Analysis using machine learning methods can be successfully applied to identify significant risk factors for oncologic outcomes.

Effect of maximum support attachment angle on intaglio surface trueness of anatomic contour monolithic prostheses manufactured by digital light processing and zirconia suspension.

STATEMENT OF PROBLEM: Support structures are essential for the quality of resin-based prostheses made by the digital light processing (DLP), but few studies have evaluated the effect of support structure on the accuracy of zirconia-based anatomic contour prostheses. PURPOSE: The purpose of this in vitro study was to evaluate the effect of maximum support attachment angle (MSA) on the intaglio surface trueness of anatomic contour prostheses made by DLP and compare the trueness of 2-unit anatomic contour prostheses with that of those produced by milling. MATERIAL AND METHODS: Anatomic contour single-unit prostheses were manufactured using DLP and a suspension with 3-mol% yttria-stabilized zirconia. Four different conditions of MSA values to the vertical axis of the object (50, 55, 60, and 65 degrees) were applied (n=10). After printing, postprocessing, and sintering, all successfully produced prostheses were evaluated for intaglio surface trueness by considering the root mean square (RMS). Using the MSA showing the highest trueness, the 2-unit prostheses made by DLP (DLP group) were compared with milled (MIL group) prostheses in terms of intaglio accuracy (n=10). One-way analysis of variance and a post hoc pairwise comparison or independent t test were used for trueness analysis (α=.05). RESULTS: Three MSA groups (50, 55, and 60 degrees) were successfully produced with significant differences between the trueness of the single-unit prostheses for the groups with different MSA values (P<.05). The highest trueness was in the 50-degree MSA group. The 2-unit prostheses of the DLP group with 50-degree MSA showed significantly lower trueness than those of the MIL group (P<.05); however, the RMS values of both groups were lower than 50 µm. CONCLUSIONS: The intaglio surface trueness of anatomic contour DLP-generated prostheses can be improved by changing the MSA. The 50-degree MSA was beneficial for the accuracy of both single-unit and 2-unit DLP-generated prostheses, produced within clinically acceptable limits.

Biological effects of gamma-ray sterilization on 3 mol% yttria-stabilized tetragonal zirconia polycrystal: An in vitro study.

STATEMENT OF PROBLEM: Selecting the sterilization method is important because sterilization can alter the surface chemistry of implant materials, including zirconia, and influence their cellular biocompatibility. Studies on the biological effects of sterilization on implant materials are lacking. PURPOSE: The purpose of this in vitro study was to evaluate the biocompatibility of gamma-ray irradiated 3 mol% yttria-stabilized tetragonal zirconia polycrystal (3Y-TZP) compared with unirradiated titanium, 3Y-TZP, and pure gold. MATERIAL AND METHODS: Disk-shaped specimens each of commercially pure grade 4 titanium, 3Y-TZP, gamma-rayed 3Y-TZP, and pure gold were prepared and evaluated for osteogenic potential by using a clonal murine cell line of immature osteoblasts derived from mice (MC3T3-E1 cells). The surface topography (n=3), chemical analysis of the disks (n=3), and cell morphology cultured on these surfaces were examined using scanning electron microscopy, confocal laser scanning microscopy, and energy dispersive spectroscopy. Cellular biocompatibility was analyzed for 1 and 3 days after seeding. Cell adhesion and spreading were evaluated using confocal laser scanning microscopy (n=3). Cell proliferation was evaluated using methyl thiazolyl tetrazolium assay (n=3). Kruskal-Wallis and Bonferroni corrections were used to evaluate the statistical significance of the intergroup differences (α=.05). RESULTS: Gamma-ray sterilization of 3Y-TZP showed significantly higher surface roughness compared with titanium and gold (P<.002). On day 1, the proliferation and adhesion of MC3T3-E1 cells cultured on gamma-rayed 3Y-TZP were significantly higher than those cultured on gold (P<.05); however, cell spreading was significantly lower than that of titanium on days 1 and 3 (P<.05). On day 3, cell proliferation of gamma-rayed 3Y-TZP was significantly lower than that of unirradiated 3Y-TZP (P<.05). Cell adhesion of gamma-rayed 3Y-TZP was slightly lower than that of zirconia and titanium but without significant difference (P>.05). CONCLUSIONS: Gamma-rayed zirconia exhibited increased surface roughness compared with titanium and significantly decreased bioactivity compared with titanium and zirconia. The use of gamma-ray sterilization on zirconia is not promising regarding biocompatibility, and the effect of this sterilization method on implant materials warrants further investigation.

Effect of CAD-CAM restorative materials and scanning aid conditions on the accuracy and time efficiency of intraoral scans.

PURPOSE: This in vitro study aimed to evaluate the effects of restorative materials and scanning aid conditions on the accuracy and time efficiency of intraoral scans. MATERIALS AND METHODS: Identical anatomic contour crowns were fabricated using the following materials: hybrid ceramic, 3 mol% yttria-stabilized tetragonal zirconia, 4 mol% yttria-partially stabilized zirconia, 5 mol% yttria-partially stabilized zirconia, cobalt-chromium (Co-Cr), resin, lithium disilicate, and feldspathic ceramic. The models were digitized and analyzed for accuracy (n = 10) under three scanning aid conditions (powder-based, liquid-based, and none). Additionally, the effect of metal restorations on the scan accuracy of other crowns was investigated. The scan time for complete arches was also recorded. One-way analysis of variance, Welch analysis of variance, and post-hoc comparison or independent t-tests were used for trueness analysis, and the F-test was used to examine precision (α = 0.05). RESULTS: Significant differences were observed in the trueness of the different restorative materials under the no-scanning aid condition (P < 0.05). In contrast, no statistically significant difference among the groups was observed with the powder- or liquid-based scanning aid. For each restorative material, the no-scanning aid condition showed significantly lower trueness than that with powder- or liquid-based scanning aids. The presence of a Co-Cr crown did not affect the trueness of other restorations in the arch. The scan time efficiency significantly increased on applying a powder- or liquid-based scanning aid. CONCLUSIONS: Using a scanning aid was effective to improve the scan accuracy of the tested restorative materials and scan time efficiency. Applying scanning aids to existing intraoral restorations can help improve prosthesis quality and reduce the need for clinical adjustment at the occlusal or proximal contacts.

Emerging roles of autophagy in hepatic tumorigenesis and therapeutic strategies in glycogen storage disease type Ia: A review.

Glycogen storage disease type Ia (GSD-Ia) is an inherited metabolic disease caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC) which plays a critical role in blood glucose homeostasis by catalyzing the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate in the terminal step of glycogenolysis and gluconeogenesis. Patients with GSD-Ia manifest life-threatening fasting hypoglycemia along with the excessive accumulation of hepatic glycogen and triglycerides which results in hepatomegaly and a risk of long-term complications such as hepatocellular adenoma and carcinoma (HCA/HCC). The etiology of HCA/HCC development in GSD-Ia, however, is unknown. Recent studies have shown that the livers in model animals of GSD-Ia display impairment of autophagy, a cellular recycling process which is critical for energy metabolism and cellular homeostasis. However, molecular mechanisms of autophagy impairment and its involvement in pathogenesis in GSD-Ia are still under investigation. Here, we summarize the latest advances for signaling pathways implicated in hepatic autophagy impairment and the roles of autophagy in hepatic tumorigenesis in GSD-Ia. In addition, recent evidence has illustrated that autophagy plays an important role in hepatic metabolism and liver-directed gene therapy mediated by recombinant adeno-associated virus (rAAV). Therefore, we highlight the possible role of hepatic autophagy in metabolic control and rAAV-mediated gene therapy for GSD-Ia. In this review, we also provide potential therapeutic strategies for GSD-Ia on the basis of molecular mechanisms underlying hepatic autophagy impairment in GSD-Ia.

Activation of tumor-promoting pathways implicated in hepatocellular adenoma/carcinoma, a long-term complication of glycogen storage disease type Ia.

Hepatocellular adenoma/carcinoma (HCA/HCC) is a long-term complication of the metabolic disorder glycogen storage disease type Ia (GSD-Ia) deficient in glucose-6-phosphatase-α (G6PC or G6Pase-α). We have shown previously that hepatic G6Pase-α deficiency leads to autophagy impairment, mitochondrial dysfunction, enhanced glycolysis, and augmented hexose monophosphate shunt, all of which can contribute to hepatocarcinogenesis. However, the mechanism underlying HCA/HCC development in GSD-Ia remains unclear. We now show that G6Pase-α deficiency-mediated hepatic autophagy impairment leads to sustained accumulation of an autophagy-specific substrate p62 which can activate tumor-promoting pathways including nuclear factor erythroid 2-related factor 2 (Nrf2) and mammalian target of rapamycin complex 1 (mTORC1). Consistently, the HCA/HCC lesions developed in the G6Pase-α-deficient livers display marked accumulation of p62 aggregates and phosphorylated p62 along with activation of Nrf2 and mTORC1 signaling. Furthermore, the HCA/HCC lesions exhibit activation of additional oncogenic pathways, ß-catenin and Yes-associated protein (YAP) which is implicated in autophagy impairment. Intriguingly, hepatic levels of glucose-6-phosphate and glycogen which are accumulated in the G6Pase-α-deficient livers were significantly lower in HCC than those in HCA. Conversely, compared to HCA, the HCC lesion display increased expression of many oncogenes and the M2 isoform of pyruvate kinase (PKM2), a glycolytic enzyme critical for aerobic glycolysis and tumorigenesis. Collectively, our data show that hepatic G6Pase-α-deficiency leads to persistent autophagy impairment and activation of multiple tumor-promoting pathways that contribute to HCA/HCC development in GSD-Ia.

Downregulation of SIRT1 signaling underlies hepatic autophagy impairment in glycogen storage disease type Ia.

A deficiency in glucose-6-phosphatase-α (G6Pase-α) in glycogen storage disease type Ia (GSD-Ia) leads to impaired glucose homeostasis and metabolic manifestations including hepatomegaly caused by increased glycogen and neutral fat accumulation. A recent report showed that G6Pase-α deficiency causes impairment in autophagy, a recycling process important for cellular metabolism. However, the molecular mechanism underlying defective autophagy is unclear. Here we show that in mice, liver-specific knockout of G6Pase-α (L-G6pc-/-) leads to downregulation of sirtuin 1 (SIRT1) signaling that activates autophagy via deacetylation of autophagy-related (ATG) proteins and forkhead box O (FoxO) family of transcriptional factors which transactivate autophagy genes. Consistently, defective autophagy in G6Pase-α-deficient liver is characterized by attenuated expressions of autophagy components, increased acetylation of ATG5 and ATG7, decreased conjugation of ATG5 and ATG12, and reduced autophagic flux. We further show that hepatic G6Pase-α deficiency results in activation of carbohydrate response element-binding protein, a lipogenic transcription factor, increased expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), a lipid regulator, and suppressed expression of PPAR-α, a master regulator of fatty acid ß-oxidation, all contributing to hepatic steatosis and downregulation of SIRT1 expression. An adenovirus vector-mediated increase in hepatic SIRT1 expression corrects autophagy defects but does not rectify metabolic abnormalities associated with G6Pase-α deficiency. Importantly, a recombinant adeno-associated virus (rAAV) vector-mediated restoration of hepatic G6Pase-α expression corrects metabolic abnormalities, restores SIRT1-FoxO signaling, and normalizes defective autophagy. Taken together, these data show that hepatic G6Pase-α deficiency-mediated down-regulation of SIRT1 signaling underlies defective hepatic autophagy in GSD-Ia.

TmAtg6 Plays an Important Role in Anti-Microbial Defense Against Listeria monocytogenes in the Mealworm, Tenebrio molitor.

Autophagy-related gene-6 (Beclin-1 in mammals) plays a pivotal role in autophagy and is involved in autophagosome formation and autolysosome maturation. In this study, we identified and characterized the autophagy-related gene-6 from Tenebrio molitor (TmAtg6) and analyzed its functional role in the survival of the insect against infection. The expression of TmAtg6 was studied using qRT-PCR for the assessment of the transcript levels at various developmental stages in the different tissues. The results showed that TmAtg6 was highly expressed at the 6-day-old pupal stage. Tissue-specific expression studies revealed that TmAtg6 was highly expressed in the hemocytes of late larvae. The induction patterns of TmAtg6 in different tissues of T. molitor larvae were analyzed by injecting Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, or Candida albicans. The intracellular Gram-positive bacteria, L. monocytogenes, solely induced the expression of TmAtg6 in hemocytes at 9 h-post-injection, whilst in the fat body and gut, bimodal expression times were observed. RNAi-mediated knockdown of the TmAtg6 transcripts, followed by a challenge with microbes, showed a significant reduction in larval survival rate against L. monocytogenes. Taken together, our results suggest that TmAtg6 plays an essential role in anti-microbial defense against intracellular bacteria.

Downregulation of pathways implicated in liver inflammation and tumorigenesis of glycogen storage disease type Ia mice receiving gene therapy.

Glycogen storage disease type Ia (GSD-Ia) is characterized by impaired glucose homeostasis and long-term risks of hepatocellular adenoma (HCA) and carcinoma (HCC). We have shown that the non-tumor-bearing (NT), recombinant adeno-associated virus (rAAV) vector-treated GSD-Ia mice (AAV-NT mice) expressing a wide range (0.9-63%) of normal hepatic glucose-6-phosphatase-α activity maintain glucose homeostasis and display physiologic features mimicking animals living under calorie restriction (CR). We now show that in AAV-NT mice, the signaling pathways of the CR mediators, AMP-activated protein kinase (AMPK) and sirtuin-1 are activated. AMPK/sirtuin-1 inhibit the activity of STAT3 (signal transducer and activator of transcription 3) and NFκB (nuclear factor κB), the pro-inflammatory and cancer-promoting transcription factors. Sirtuin-1 also inhibits cancer metastasis via increasing the expression of E-cadherin, a tumor suppressor, and decreasing the expression of mesenchymal markers. Consistently, in AAV-NT mice, hepatic levels of active STAT3 and NFκB-p65 were reduced as were expression of mesenchymal markers, STAT3 targets, NFκB targets and ß-catenin targets, all of which were consistent with the promotion of tumorigenesis. AAV-NT mice also expressed increased levels of E-cadherin and fibroblast growth factor 21 (FGF21), targets of sirtuin-1, and ß-klotho, which can acts as a tumor suppressor. Importantly, treating AAV-NT mice with a sirtuin-1 inhibitor markedly reversed many of the observed anti-inflammatory/anti-tumorigenic signaling pathways. In summary, activation of hepatic AMPK/sirtuin-1 and FGF21/ß-klotho signaling pathways combined with down-regulation of STAT3/NFκB-mediated inflammatory and tumorigenic signaling pathways can explain the absence of hepatic tumors in AAV-NT mice.

Liver-directed gene therapy for murine glycogen storage disease type Ib.

Glycogen storage disease type-Ib (GSD-Ib), deficient in the glucose-6-phosphate transporter (G6PT), is characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of hepatocellular adenoma (HCA). We examined the efficacy of G6PT gene therapy in G6pt-/- mice using recombinant adeno-associated virus (rAAV) vectors, directed by either the G6PC or the G6PT promoter/enhancer. Both vectors corrected hepatic G6PT deficiency in murine GSD-Ib but the G6PC promoter/enhancer was more efficacious. Over a 78-week study, using dose titration of the rAAV vectors, we showed that G6pt-/- mice expressing 3-62% of normal hepatic G6PT activity exhibited a normalized liver phenotype. Two of the 12 mice expressing < 6% of normal hepatic G6PT activity developed HCA. All treated mice were leaner and more sensitive to insulin than wild-type mice. Mice expressing 3-22% of normal hepatic G6PT activity exhibited higher insulin sensitivity than mice expressing 44-62%. The levels of insulin sensitivity correlated with the magnitudes of hepatic carbohydrate response element binding protein signaling activation. In summary, we established the threshold of hepatic G6PT activity required to prevent tumor formation and showed that mice expressing 3-62% of normal hepatic G6PT activity maintained glucose homeostasis and were protected against age-related obesity and insulin resistance.

An evolutionary approach to optimizing glucose-6-phosphatase-α enzymatic activity for gene therapy of glycogen storage disease type Ia.

Glycogen storage disease type-Ia (GSD-Ia), caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), is characterized by impaired glucose homeostasis with a hallmark hypoglycemia, following a short fast. We have shown that G6pc-deficient (G6pc-/-) mice treated with recombinant adeno-associated virus (rAAV) vectors expressing either wild-type (WT) (rAAV-hG6PC-WT) or codon-optimized (co) (rAAV-co-hG6PC) human (h) G6Pase-α maintain glucose homeostasis if they restore ≥3% of normal hepatic G6Pase-α activity. The co vector, which has a higher potency, is currently being used in a phase I/II clinical trial for human GSD-Ia (NCT03517085). While routinely used in clinical therapies, co vectors may not always be optimal. Codon-optimization can impact RNA secondary structure, change RNA/DNA protein-binding sites, affect protein conformation and function, and alter posttranscriptional modifications that may reduce potency or efficacy. We therefore sought to develop alternative approaches to increase the potency of the G6PC gene transfer vectors. Using an evolutionary sequence analysis, we identified a Ser-298 to Cys-298 substitution naturally found in canine, mouse, rat, and several primate G6Pase-α isozymes, that when incorporated into the WT hG6Pase-α sequence, markedly enhanced enzymatic activity. Using G6pc-/- mice, we show that the efficacy of the rAAV-hG6PC-S298C vector was 3-fold higher than that of the rAAV-hG6PC-WT vector. The rAAV-hG6PC-S298C vector with increased efficacy, that minimizes the potential problems associated with codon-optimization, offers a valuable vector for clinical translation in human GSD-Ia.

Gene therapy prevents hepatic tumor initiation in murine glycogen storage disease type Ia at the tumor-developing stage.

Hepatocellular adenoma/carcinoma (HCA/HCC) is a long-term complication of glycogen storage disease type-Ia (GSD-Ia), which is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), a key enzyme in gluconeogenesis. Currently, there is no therapy to address HCA/HCC in GSD-Ia. We have previously shown that a recombinant adeno-associated virus (rAAV) vector-mediated G6PC gene transfer to 2-week-old G6pc-/- mice prevents HCA development. However, it remains unclear whether G6PC gene transfer at the tumor developing stage of GSD-Ia can prevent tumor initiation or abrogate the pre-existing tumors. Using liver-specific G6pc-knockout (L-G6pc-/-) mice that develop HCA/HCC, we now show that treating the mice at the tumor-developing stage with rAAV-G6PC restores hepatic G6Pase-α expression, normalizes glucose homeostasis, and prevents de novo HCA/HCC development. The rAAV-G6PC treatment also normalizes defective hepatic autophagy and corrects metabolic abnormalities in the nontumor liver tissues of both tumor-free and tumor-bearing mice. However, gene therapy cannot restore G6Pase-α expression in the HCA/HCC lesions and fails to abrogate any pre-existing tumors. We show that the expression of 11 ß-hydroxysteroid dehydrogenase type-1 that mediates local glucocorticoid activation is downregulated in HCA/HCC lesions, leading to impairment in glucocorticoid signaling critical for gluconeogenesis activation. This suggests that local glucocorticoid action downregulation in the HCA/HCC lesions may suppress gene therapy mediated G6Pase-α restoration. Collectively, our data show that rAAV-mediated gene therapy can prevent de novo HCA/HCC development in L-G6pc-/- mice at the tumor developing stage, but it cannot reduce any pre-existing tumor burden.

PRMT1 negatively regulates activation-induced cell death in macrophages by arginine methylation of GAPDH.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is implicated in cell death in addition to a role as a glycolytic enzyme. In particular, when cells are exposed to cellular stressors involving nitric oxide (NO) production, GAPDH can undergo NO-induced S-nitrosylation and S-nitrosylated GAPDH has been shown to elicit apoptosis. However, the mechanism underlying the regulation of the pro-apoptotic function of GAPDH remains unclear. Here, we found that protein arginine methyltransferase 1 (PRMT1) mediated arginine methylation of GAPDH in primary bone marrow-derived macrophages in a NO-dependent manner. Moreover, PRMT1 inhibited S-nitrosylation of GAPDH as well as its binding to SIAH1, thereby reducing the nuclear translocation of GAPDH in lipopolysaccharide (LPS)/interferon (IFN)-γ-activated macrophages. Furthermore, depletion of PRMT1 expression by RNA interference potentiated LPS/IFN-γ-induced apoptosis in macrophages. Taken together, our results suggest that PRMT1 has a previously unrecognized function to inhibit activation-induced cell death of macrophages through arginine methylation of GAPDH.

Hepatic glucose-6-phosphatase-α deficiency leads to metabolic reprogramming in glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), a key enzyme in endogenous glucose production. This autosomal recessive disorder is characterized by impaired glucose homeostasis and long-term complications of hepatocellular adenoma/carcinoma (HCA/HCC). We have shown that hepatic G6Pase-α deficiency-mediated steatosis leads to defective autophagy that is frequently associated with carcinogenesis. We now show that hepatic G6Pase-α deficiency also leads to enhancement of hepatic glycolysis and hexose monophosphate shunt (HMS) that can contribute to hepatocarcinogenesis. The enhanced hepatic glycolysis is reflected by increased lactate accumulation, increased expression of many glycolytic enzymes, and elevated expression of c-Myc that stimulates glycolysis. The increased HMS is reflected by increased glucose-6-phosphate dehydrogenase activity and elevated production of NADPH and the reduced glutathione. We have previously shown that restoration of hepatic G6Pase-α expression in G6Pase-α-deficient liver corrects metabolic abnormalities, normalizes autophagy, and prevents HCA/HCC development in GSD-Ia. We now show that restoration of hepatic G6Pase-α expression normalizes both glycolysis and HMS in GSD-Ia. Moreover, the HCA/HCC lesions in L-G6pc-/- mice exhibit elevated levels of hexokinase 2 (HK2) and the M2 isoform of pyruvate kinase (PKM2) which play an important role in aerobic glycolysis and cancer cell proliferation. Taken together, hepatic G6Pase-α deficiency causes metabolic reprogramming, leading to enhanced glycolysis and elevated HMS that along with impaired autophagy can contribute to HCA/HCC development in GSD-Ia.

Sirtuin signaling controls mitochondrial function in glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) deficient in glucose-6-phosphatase-α (G6Pase-α) is a metabolic disorder characterized by impaired glucose homeostasis and a long-term complication of hepatocellular adenoma/carcinoma (HCA/HCC). Mitochondrial dysfunction has been implicated in GSD-Ia but the underlying mechanism and its contribution to HCA/HCC development remain unclear. We have shown that hepatic G6Pase-α deficiency leads to downregulation of sirtuin 1 (SIRT1) signaling that underlies defective hepatic autophagy in GSD-Ia. SIRT1 is a NAD+-dependent deacetylase that can deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), a master regulator of mitochondrial integrity, biogenesis, and function. We hypothesized that downregulation of hepatic SIRT1 signaling in G6Pase-α-deficient livers impairs PGC-1α activity, leading to mitochondrial dysfunction. Here we show that the G6Pase-α-deficient livers display defective PGC-1α signaling, reduced numbers of functional mitochondria, and impaired oxidative phosphorylation. Overexpression of hepatic SIRT1 restores PGC-1α activity, normalizes the expression of electron transport chain components, and increases mitochondrial complex IV activity. We have previously shown that restoration of hepatic G6Pase-α expression normalized SIRT1 signaling. We now show that restoration of hepatic G6Pase-α expression also restores PGC-1α activity and mitochondrial function. Finally, we show that HCA/HCC lesions found in G6Pase-α-deficient livers contain marked mitochondrial and oxidative DNA damage. Taken together, our study shows that downregulation of hepatic SIRT1/PGC-1α signaling underlies mitochondrial dysfunction and that oxidative DNA damage incurred by damaged mitochondria may contribute to HCA/HCC development in GSD-Ia.

Molecular biology and gene therapy for glycogen storage disease type Ib.

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the ubiquitously expressed glucose-6-phosphate (G6P) transporter (G6PT or SLC37A4). The primary function of G6PT is to translocate G6P from the cytoplasm into the lumen of the endoplasmic reticulum (ER). Inside the ER, G6P is hydrolyzed to glucose and phosphate by either the liver/kidney/intestine-restricted glucose-6-phosphatase-α (G6Pase-α) or the ubiquitously expressed G6Pase-ß. A deficiency in G6Pase-α causes GSD type Ia (GSD-Ia) and a deficiency in G6Pase-ß causes GSD-I-related syndrome (GSD-Irs). In gluconeogenic organs, functional coupling of G6PT and G6Pase-α is required to maintain interprandial blood glucose homeostasis. In myeloid tissues, functional coupling of G6PT and G6Pase-ß is required to maintain neutrophil homeostasis. Accordingly, GSD-Ib is a metabolic and immune disorder, manifesting impaired glucose homeostasis, neutropenia, and neutrophil dysfunction. A G6pt knockout mouse model is being exploited to delineate the pathophysiology of GSD-Ib and develop new clinical treatment options, including gene therapy. The safety and efficacy of several G6PT-expressing recombinant adeno-associated virus pseudotype 2/8 vectors have been examined in murine GSD-Ib. The results demonstrate that the liver-directed gene transfer and expression safely corrects metabolic abnormalities and prevents hepatocellular adenoma (HCA) development. However, a second vector system may be required to correct myeloid and renal dysfunction in GSD-Ib. These findings are paving the way to a safe and efficacious gene therapy for entering clinical trials.

Mice expressing reduced levels of hepatic glucose-6-phosphatase-α activity do not develop age-related insulin resistance or obesity.

Glycogen storage disease type-Ia (GSD-Ia) is caused by a lack of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. We have shown that gene therapy mediated by a recombinant adeno-associated virus (rAAV) vector expressing human G6Pase-α normalizes blood glucose homeostasis in the global G6pc knockout (G6pc(-/-)) mice for 70-90 weeks. The treated G6pc(-/-) mice expressing 3-63% of normal hepatic G6Pase-α activity (AAV mice) produce endogenous hepatic glucose levels 61-68% of wild-type littermates, have a leaner phenotype and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by hepatic carbohydrate response element binding protein that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including the NADH shuttle systems, NAD(+) concentrations and the AMP-activated protein kinase/sirtuin 1/peroxisome proliferator-activated receptor-γ coactivator 1α pathway are upregulated in the livers of AAV mice. The finding that partial restoration of hepatic G6Pase-α activity in GSD-Ia mice not only attenuates the phenotype of hepatic G6Pase-α deficiency but also prevents the development of age-related obesity and insulin resistance seen in wild-type mice may suggest relevance of the G6Pase-α enzyme to obesity and diabetes.

Glycogen storage disease type Ia mice with less than 2% of normal hepatic glucose-6-phosphatase-α activity restored are at risk of developing hepatic tumors.

Glycogen storage disease type Ia (GSD-Ia), characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA) and carcinoma (HCC), is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). We have previously shown that G6pc-/- mice receiving gene transfer mediated by rAAV-G6PC, a recombinant adeno-associated virus (rAAV) vector expressing G6Pase-α, and expressing 3-63% of normal hepatic G6Pase-α activity maintain glucose homeostasis and do not develop HCA/HCC. However, the threshold of hepatic G6Pase-α activity required to prevent tumor formation remained unknown. In this study, we constructed rAAV-co-G6PC, a rAAV vector expressing a codon-optimized (co) G6Pase-α and showed that rAAV-co-G6PC was more efficacious than rAAV-G6PC in directing hepatic G6Pase-α expression. Over an 88-week study, we showed that both rAAV-G6PC- and rAAV-co-G6PC-treated G6pc-/- mice expressing 3-33% of normal hepatic G6Pase-α activity (AAV mice) maintained glucose homeostasis, lacked HCA/HCC, and were protected against age-related obesity and insulin resistance. Of the eleven rAAV-G6PC/rAAV-co-G6PC-treated G6pc-/- mice harboring 0.9-2.4% of normal hepatic G6Pase-α activity (AAV-low mice), 3 expressing 0.9-1.3% of normal hepatic G6Pase-α activity developed HCA/HCC, while 8 did not (AAV-low-NT). Finally, we showed that the AAV-low-NT mice exhibited a phenotype indistinguishable from that of AAV mice expressing ≥3% of normal hepatic G6Pase-α activity. The results establish the threshold of hepatic G6Pase-α activity required to prevent HCA/HCC and show that GSD-Ia mice harboring <2% of normal hepatic G6Pase-α activity are at risk of tumor development.

Deep learning-designed implant-supported posterior crowns: Assessing time efficiency, tooth morphology, emergence profile, occlusion, and proximal contacts.

OBJECTIVES: To compare implant supported crowns (ISCs) designed using deep learning (DL) software with those designed by a technician using conventional computer-aided design software. METHODS: Twenty resin-based partially edentulous casts (maxillary and mandibular) used for fabricating ISCs were evaluated retrospectively. ISCs were designed using a DL-based method with no modification of the as-generated outcome (DB), a DL-based method with further optimization by a dental technician (DM), and a conventional computer-aided design method by a technician (NC). Time efficiency, crown contour, occlusal table area, cusp angle, cusp height, emergence profile angle, occlusal contacts, and proximal contacts were compared among groups. Depending on the distribution of measured data, various statistical methods were used for comparative analyses with a significance level of 0.05. RESULTS: ISCs in the DB group showed a significantly higher efficiency than those in the DM and NC groups (P ≤ 0.001). ISCs in the DM group exhibited significantly smaller volume deviations than those in the DB group when superimposed on ISCs in the NC group (DB-NC vs. DM-NC pairs, P ≤ 0.008). Except for the number and intensity of occlusal contacts (P ≤ 0.004), ISCs in the DB and DM groups had occlusal table areas, cusp angles, cusp heights, proximal contact intensities, and emergence profile angles similar to those in the NC group (P ≥ 0.157). CONCLUSIONS: A DL-based method can be beneficial for designing posterior ISCs in terms of time efficiency, occlusal table area, cusp angle, cusp height, proximal contact, and emergence profile, similar to the conventional human-based method. CLINICAL SIGNIFICANCE: A deep learning-based design method can achieve clinically acceptable functional properties of posterior ISCs. However, further optimization by a technician could improve specific outcomes, such as the crown contour or emergence profile angle.

Antimicrobial activity, surface properties, and cytotoxicity of microencapsulated phytochemicals incorporated into three-dimensionally printable dental polymers.

OBJECTIVES: This study aimed to investigate the antimicrobial properties of three dimensionally-printed dental polymers (3DPs) incorporated with microencapsulated phytochemicals (MPs) and to assess their surface characteristics and cytotoxicity. METHODS: MPs derived from phytoncide oil and their specific chemical components were introduced into suspensions of three microbial species: Streptococcus gordonii, Streptococcus oralis, and Candida albicans. Optical density was measured to determine the microbial growth in the presence of MPs for testing their antimicrobial activity. MPs at 5% (w/w) were mixed with dental polymers and dispersants to 3DP discs. These microbial species were then seeded onto the discs and incubated for 24 h. The antibacterial and antifungal activities of MP-containing 3DPs were evaluated by counting the colony-forming units (n = 3). The biofilm formation on the 3DP was assessed by crystal violet staining assay (n = 3). Microbial viability was determined using a live-dead staining and CLSM observation (n = 3). Surface roughness and water contact angle were assessed (n = 10). Cytotoxicity of MP-containing 3DPs for human gingival fibroblast was evaluated by MTT assay. RESULTS: MPs, particularly (-)-α-pinene, suppressed the growth of all tested microbial species. MP-containing 3DPs significantly reduced the colony count (P ≤ 0.001) and biofilm formation (P ≤ 0.009), of all tested microbial species. Both surface roughness (P < 0.001) and water contact angle (P < 0.001) increased. The cytotoxicity remained unchanged after incorporating MPs to the 3DPs (P = 0.310). CONCLUSIONS: MPs effectively controlled the microbial growth on 3DPs as evidenced by the colony count, biofilm formation, and cell viability. Although MPs modified the surface characteristics, they did not influence the cytotoxicity of 3DPs. CLINICAL SIGNIFICANCE: Integration of MPs into 3DPs could produce dental prostheses or appliances with antimicrobial properties. This approach not only provides a proactive solution to reduce the risk of oral biofilm-related infection but also ensures the safety and biocompatibility of the material, thereby improving dental care.