Topographical and crystalline change on surface by sandblasting improve flexural and shear bond strength of niobia-modified yttria-stabilized tetragonal zirconia polycrystal.

This study aimed to investigate the effects of sandblasting on the physical properties and bond strength of two types of translucent zirconia: niobium-oxide-containing yttria-stabilized tetragonal zirconia polycrystals ((Y, Nb)-TZP) and 5 mol% yttria-partially stabilized zirconia (5Y-PSZ). Fully sintered disc specimens were either sandblasted with 125 µm alumina particles or left as-sintered. Surface roughness, crystal phase compositions, and surface morphology were explored. Biaxial flexural strength (n=10) and shear bond strength (SBS) (n=12) were evaluated, including thermocycling conditions. Results indicated a decrease in flexural strength of 5Y-PSZ from 601 to 303 MPa upon sandblasting, while (Y, Nb)-TZP improved from 458 to 544 MPa. Both materials significantly increased SBS after sandblasting (p<0.001). After thermocycling, (Y, Nb)-TZP maintained superior SBS (14.3 MPa) compared to 5Y-PSZ (11.3 MPa) (p<0.001). The study concludes that (Y, Nb)-TZP is preferable for sandblasting applications, particularly for achieving durable bonding without compromising flexural strength.

Tooth morphology, internal fit, occlusion and proximal contacts of dental crowns designed by deep learning-based dental software: A comparative study.

OBJECTIVES: This study compared the tooth morphology, internal fit, occlusion, and proximal contacts of dental crowns automatically generated via two deep learning (DL)-based dental software systems with those manually designed by an experienced dental technician using conventional software. METHODS: Thirty partial arch scans of prepared posterior teeth were used. The crowns were designed using two DL-based methods (AA and AD) and a technician-based method (NC). The crown design outcomes were three-dimensionally compared, focusing on tooth morphology, internal fit, occlusion, and proximal contacts, by calculating the geometric relationship. Statistical analysis utilized the independent t-test, Mann-Whitney test, one-way ANOVA, and Kruskal-Wallis test with post hoc pairwise comparisons (α = 0.05). RESULTS: The AA and AD groups, with the NC group as a reference, exhibited no significant tooth morphology discrepancies across entire external or occlusal surfaces. The AD group exhibited higher root mean square and positive average values on the axial surface (P < .05). The AD and NC groups exhibited a better internal fit than the AA group (P < .001). The cusp angles were similar across all groups (P = .065). The NC group yielded more occlusal contact points than the AD group (P = .006). Occlusal and proximal contact intensities varied among the groups (both P < .001). CONCLUSIONS: Crowns designed by using both DL-based software programs exhibited similar morphologies on the occlusal and axial surfaces; however, they differed in internal fit, occlusion, and proximal contacts. Their overall performance was clinically comparable to that of the technician-based method in terms of the internal fit and number of occlusal contact points. CLINICAL SIGNIFICANCE: DL-based dental software for crown design can streamline the digital workflow in restorative dentistry, ensuring clinically-acceptable outcomes on tooth morphology, internal fit, occlusion, and proximal contacts. It can minimize the necessity of additional design optimization by dental technician.

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.

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.

Time efficiency, occlusal morphology, and internal fit of anatomic contour crowns designed by dental software powered by generative adversarial network: A comparative study.

OBJECTIVES: To evaluate the time efficiency, occlusal morphology, and internal fit of dental crowns designed using generative adversarial network (GAN)-based dental software compared to conventional dental software. METHODS: Thirty datasets of partial arch scans for prepared posterior teeth were analyzed. Each crown was designed on each abutment using GAN-based software (AI) and conventional dental software (non-AI). The AI and non-AI groups were compared in terms of time efficiency by measuring the elapsed work time. The difference in the occlusal morphology of the crowns before and after design optimization and the internal fit of the crown to the prepared abutment were also evaluated by superimposition for each software. Data were analyzed using independent t tests or Mann-Whitney test with statistical significance (α=.05). RESULTS: The working time was significantly less for the AI group than the non-AI group at T1, T5, and T6 (P≤.043). The working time with AI was significantly shorter at T1, T3, T5, and T6 for the intraoral scan (P≤.036). Only at T2 (P≤.001) did the cast scan show a significant difference between the two groups. The crowns in the AI group showed less deviation in occlusal morphology and significantly better internal fit to the abutment than those in the non-AI group (both P<.001). CONCLUSIONS: Crowns designed by AI software showed improved outcomes than that designed by non-AI software, in terms of time efficiency, difference in occlusal morphology, and internal fit. CLINICAL SIGNIFICANCE: The GAN-based software showed better time efficiency and less deviation in occlusal morphology during the design process than the conventional software, suggesting a higher probability of optimized outcomes of crown design.

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 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.

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.

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.

A method to implement the electrode-entropy differentiation for lithium batteries.

This work presents the method and apparatus for the reproduction of the electrode-entropy differentiation approach which provides a tool to nondesctructively determine the contribution of each electrode to the battery total entropy by performing a comparative study with another battery which shares the same composition for only one of its electrodes (semi-similar types). The 2 cells must go through the same aging process so the proposed method can be applied as follow. Firstly, an optional pre-processing step is performed, followed by linear regression between capacity loss and entropy evolution over the full SOC range. Then, a correlation is computed for each SOC value between the entropy evolution of the two cells. The result of that correlation shows at which SOC the evolution of the two cells is similar. Postulating that half-cells of the same chemistry going under the same aging conditions age the same way, the common-mode highlighted by the correlation is the SOC-domain at which the entropy of the common half-cell is dominating. This method allows improves prior art by removing the need for custom-made half-cells of the same battery composition. The feasibility of the electrode-entropy differentiation methods is postulated, and the method is detailed. A validation study is performed on a set of batteries and the method feasibility is confirmed by the results obtained. This paper:•Demonstrates the feasibility of extracting the entropy from the comparative study between the subject cell and a reference.•Includes a validation test.•Suggest application of the method in the field of electrodes diagnosis.

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.

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.

Liver Glycogen Phosphorylase Deficiency Leads to Profibrogenic Phenotype in a Murine Model of Glycogen Storage Disease Type VI.

Mutations in the liver glycogen phosphorylase (Pygl) gene are associated with the diagnosis of glycogen storage disease type VI (GSD-VI). To understand the pathogenesis of GSD-VI, we generated a mouse model with Pygl deficiency (Pygl -/-). Pygl -/- mice exhibit hepatomegaly, excessive hepatic glycogen accumulation, and low hepatic free glucose along with lower fasting blood glucose levels and elevated blood ketone bodies. Hepatic glycogen accumulation in Pygl -/- mice increases with age. Masson's trichrome and picrosirius red staining revealed minimal to mild collagen deposition in periportal, subcapsular, and/or perisinusoidal areas in the livers of old Pygl -/- mice (>40 weeks). Consistently, immunohistochemical analysis showed the number of cells positive for alpha smooth muscle actin (α-SMA), a marker of activated hepatic stellate cells, was increased in the livers of old Pygl -/- mice compared with those of age-matched wild-type (WT) mice. Furthermore, old Pygl -/- mice had inflammatory infiltrates associated with hepatic vessels in their livers along with up-regulated hepatic messenger RNA levels of C-C chemokine ligand 5 (Ccl5/Rantes) and monocyte chemoattractant protein 1 (Mcp-1), indicating inflammation, while age-matched WT mice did not. Serum levels of aspartate aminotransferase and alanine aminotransferase were elevated in old Pygl -/- mice, indicating liver damage. Conclusion: Pygl deficiency results in progressive accumulation of hepatic glycogen with age and liver damage, inflammation, and collagen deposition, which can increase the risk of liver fibrosis. Collectively, the Pygl-deficient mouse recapitulates clinical features in patients with GSD-VI and provides a model to elucidate the mechanisms underlying hepatic complications associated with defective glycogen metabolism.

Trueness of the Inner Surface of Monolithic Crowns Fabricated by Milling of a Fully Sintered (Y, Nb)-TZP Block in Chairside CAD-CAM System for Single-visit Dentistry.

A single-visit zirconia restoration can be easily achieved if direct milling of a fully sintered zirconia block can be performed without much effort. However, no studies have yet been reported regarding the evaluation of the trueness of crown fabricated from chairside-milling of a fully sintered zirconia block in the chairside computer-aided design and computer-aided manufacturing (CAD-CAM) system for single-visit dentistry. This in vitro study aimed to evaluate the trueness of crowns fabricated by milling a fully sintered zirconia block in the chairside CAD-CAM system and investigate the clinical implications for single-visit chairside restoration. Crowns were fabricated either by chairside-milling a fully sintered block of niobium oxide containing yttria-stabilized tetragonal zirconia polycrystals ((Y, Nb)-TZP) without the sintering process (n = 12) in a chairside single-visit dentistry system (Chairside group) or by laboratory-milling a partially sintered 3 mol% block of yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP) followed by the sintering process (n = 12) in a conventional laboratory system (Labside group). Crown fabrication time, milling tool diameter and the trueness of each crown were evaluated. All trueness values of both groups were within the clinically acceptable range, although a significant difference between the Chairside (43.0 ± 3.67 µm) and Labside groups (37.4 ± 2.41 µm) was observed (P < 0.05). Mean fabrication time was 0.52 h and 1.42 h for Chairside and Labside groups, respectively. A decrease in the tool diameter was observed for the Chairside group.

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.

Molecular Cloning and Effects of Tm14-3-3ζ-Silencing on Larval Survivability Against E. coli and C. albicans in Tenebrio molitor.

The 14-3-3 family of proteins performs key regulatory functions in phosphorylation-dependent signaling pathways including cell survival and proliferation, apoptosis, regulation of chromatin structure and autophagy. In this study, the zeta isoform of 14-3-3 proteins (designated as Tm14-3-3ζ) was identified from the expressed sequence tags (ESTs) and RNA sequencing (RNA-Seq) database of the coleopteran pest, Tenebrio molitor. Tm14-3-3ζ messenger RNA (mRNA) is expressed at higher levels in the immune organs of the larval and adult stages of the insect and exhibit almost five-fold induction within 3 h post-infection of the larvae with Escherichia coli and Candida albicans. To investigate the biological function of Tm14-3-3ζ, a peptide-based Tm14-3-3ζ polyclonal antibody was generated in rabbit and the specificity was confirmed using Western blot analysis. Immunostaining and confocal microscopic analyses indicate that Tm14-3-3ζ is mainly expressed in the membranes of midgut epithelial cells, the nuclei of fat body and the cytosol of hemocytes. Gene silencing of Tm14-3-3ζ increases mortality of the larvae at 7 days post-infection with E. coli and C. albicans. Our findings demonstrate that 14-3-3ζ in T. molitor is essential in the host defense mechanisms against bacteria and fungi.

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.

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.

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.