Galactose-1-phosphate inhibits cytochrome c oxidase and causes mitochondrial dysfunction in classic galactosemia.

Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid - that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia.

Sphingolipid depletion suppresses UPR activation and promotes galactose hypersensitivity in yeast models of classic galactosemia.

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations on the GALT gene, which encodes the Leloir pathway enzyme galactose-1-phosphate uridyltransferase. Previous studies have shown that the endoplasmic reticulum unfolded protein response (UPR) is relevant to galactosemia, but the molecular mechanism behind the endoplasmic reticulum stress that triggers this response remains elusive. In the present work, we show that the activation of the UPR in yeast models of galactosemia does not depend on the binding of unfolded proteins to the ER stress sensor protein Ire1p since the protein domain responsible for unfolded protein binding to Ire1p is not necessary for UPR activation. Interestingly, myriocin - an inhibitor of the de novo sphingolipid synthesis pathway - inhibits UPR activation and causes galactose hypersensitivity in these models, indicating that myriocin-mediated sphingolipid depletion impairs yeast adaptation to galactose toxicity. Supporting the interpretation that the effects observed after myriocin treatment were due to a reduction in sphingolipid levels, the addition of phytosphingosine to the culture medium reverses all myriocin effects tested. Surprisingly, constitutively active UPR signaling did not prevent myriocin-induced galactose hypersensitivity suggesting multiple roles for sphingolipids in the adaptation of yeast cells to galactose toxicity. Therefore, we conclude that sphingolipid homeostasis has an important role in UPR activation and cellular adaptation in yeast models of galactosemia, highlighting the possible role of lipid metabolism in the pathophysiology of this disease.

Clostridioides difficile Toxin A-Induced Wnt/ß-Catenin Pathway Inhibition Is Mediated by Rac1 Glucosylation.

Clostridioides difficile toxin A (TcdA) has been shown to inhibit cellular Wnt signaling, the major driving force behind the proliferation of epithelial cells in colonic crypts, likely through the inhibition of ß-catenin nuclear translocation. Herein, we aimed to advance the understanding of this mechanism by replicating the findings in vivo and by investigating the specific role of Rac1, a member of the Rho GTPase family, on the inhibition of the Wnt-induced ß-catenin nuclear translocation triggered by TcdA. To investigate the effects of TcdA on the Wnt/ß-catenin pathway in vivo, we injected the ileal loops of C57BL/6 mice with TcdA [phosphate-buffered saline (PBS) as the control] to induce C. difficile disease-like ileitis. After 4 h post-injection, we obtained ileum tissue samples to assess Wnt signaling activation and cell proliferation through Western blotting, immunohistochemistry, and qPCR. To assess the role of Rac1 on Wnt signaling inhibition by TcdA, we transfected rat intestinal epithelial cells (IEC-6) with either a constitutively active Rac1 plasmid (pcDNA3-EGFP-Rac1-Q61L) or an empty vector, which served as the control. We incubated these cells with Wnt3a-conditioned medium (Wnt3a-CM) to induce Wnt/ß-catenin pathway activation, and then challenged the cells with TcdA. We assessed Wnt signaling activation in vitro with TOP/FOPflash luciferase assays, determined nuclear ß-catenin translocation by immunofluorescence, measured cyclin D1 protein expression by Western blotting, and quantified cell proliferation by Ki67 immunostaining. In vivo, TcdA decreased ß-catenin, cyclin D1, and cMYC expression and inhibited the translocation of ß-catenin into the nucleus in the ileum epithelial cells. In addition, TcdA suppressed cell proliferation and increased Wnt3a expression, but did not alter Rac1 gene expression in the ileum tissue. In vitro, constitutively active Rac1 prevented Wnt signaling inhibition by enabling the ß-catenin nuclear translocation that had been blocked by TcdA. Our results show that TcdA inhibits Wnt/ß-catenin pathway in vivo and demonstrate that this inhibition is likely caused by a Rac1-mediated mechanism.

Optical and Electrochemical Applications of Li-Doped NiO Nanostructures Synthesized via Facile Microwave Technique.

Nanostructured NiO and Li-ion doped NiO have been synthesized via a facile microwave technique and simulated using the first principle method. The effects of microwaves on the morphology of the nanostructures have been studied by Field Emission Spectroscopy. X-ray diffraction studies confirm the nanosize of the particles and favoured orientations along the (111), (200) and (220) planes revealing the cubic structure. The optical band gap decreases from 3.3 eV (pure NiO) to 3.17 eV (NiO doped with 1% Li). Further, computational simulations have been performed to understand the optical behaviour of the synthesized nanoparticles. The optical properties of the doped materials exhibit violet, blue and green emissions, as evaluated using photoluminescence (PL) spectroscopy. In the presence of Li-ions, NiO nanoparticles exhibit enhanced electrical capacities and better cyclability. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) results show that with 1% Li as dopant, there is a marked improvement in the reversibility and the conductance value of NiO. The results are encouraging as the synthesized nanoparticles stand a better chance of being used as an active material for electrochromic, electro-optic and supercapacitor applications.

Evaluation of in vitro Antifungal Activity of Xylosma prockia (Turcz.) Turcz. (Salicaceae) Leaves Against Cryptococcus spp.

Cryptococcus species are responsible for important systemic mycosis and are estimated to cause millions of new cases annually. The available therapy is limited due to the high toxicity and the increasing rates of yeast resistance to antifungal drugs. Popularly known as "sucará," Xylosma prockia (Turcz.) Turcz. (Salicaceae) is a native plant from Brazil with little information on its pharmacological potential. In this work, we evaluated in vitro anticryptococcal effects of the leaf ethanolic extract of X. prockia and its fractions against Cryptococcus gattii and Cryptococcus neoformans. We also evaluated phenotypic alterations caused by ethyl acetate fraction (EAF) (chosen according to its biological results). The liquid chromatography-mass spectrometry (LC-MS) analysis of EAF demonstrated the presence of phenolic metabolites that belong to three structurally related groups as majority compounds: caffeoylquinic acid, coumaroyl-glucoside, and caffeoyl-glucoside/deoxyhexosyl-caffeoyl glucoside derivatives. The minimum inhibitory concentration (MIC) values against C. gattii and C. neoformans ranged from 8 to 64 mg/L and from 0.5 to 8 mg/L, for ethanolic extract and EAF, respectively. The EAF triggered an oxidative burst and promoted lipid peroxidation. EAF also induced a reduction of ergosterol content in the pathogen cell membrane. These effects were not associated with alterations in the cell surface charge or in the thermodynamic fingerprint of the molecular interaction between EAF and the yeasts evaluated. Cytotoxic experiments with peripheral blood mononuclear cells (PBMCs) demonstrated that EAF was more selective for yeasts than was PBMCs. The results may provide evidence that X. prockia leaf extract might indeed be a potential source of antifungal agents.

Biodentine and MTA modulate immunoinflammatory response favoring bone formation in sealing of furcation perforations in rat molars.

OBJECTIVES: Evaluate the tissue reaction of periodontium subjacent to furcation perforations in rat molars sealed with Biodentine or mineral trioxide aggregate (MTA). MATERIALS AND METHODS: The pulp chamber floor of right upper first molars of 60 rats was perforated and filled with Biodentine, MTA, or cotton pellet (sham); the left first molars were used as control. After 7, 15, 30, and 60 days, maxillary fragments were processed for paraffin-embedding. The periodontal space (PS), volume density of inflammatory cells (VvIC) and fibroblasts (VvFb), number of osteoclasts, and collagen content were obtained. Interleukin-6 (IL-6) and osterix (osteoblast marker) were detected by immunohistochemistry. The data were submitted to ANOVA and Tukey's test (p ≤ 0.05). RESULTS: At 7 days, high values in VvIC, IL-6-immunolabeled cells, and osteoclasts were accompanied by reduced collagen content in enlarged PS of experimental groups. At all periods, VvIC, number of osteoclasts and IL-6, and PS were higher in sham than in Biodentine and MTA (p < 0.0001). From 7 to 60 days, significant reduction in VvIC, IL-6 immunoexpression, and osteoclasts was accompanied by significant increase in VvFb, osteoblasts, and collagen in Biodentine and MTA groups. At 60 days, significant differences in VvIC, PS, IL-6, osteoclasts, and osteoblasts were not found between Biodentine and MTA. Significant differences in the osteoclast number were not observed among Biodentine, MTA, and control groups while osteoblasts number was higher in Biodentine and MTA groups. CONCLUSIONS: Despite the initial inflammatory reaction and bone resorption, the sealing of furcation perforations with Biodentine and MTA favors the repair of periodontal tissues. CLINICAL RELEVANCE: Biodentine and MTA exhibit potential as repair material in the treatment of furcation perforations.

Tissue Response and Immunoexpression of Interleukin 6 Promoted by Tricalcium Silicate-based Repair Materials after Subcutaneous Implantation in Rats.

INTRODUCTION: The aim of the present study was to evaluate the inflammatory response induced by experimental tricalcium silicate cement with 20% zirconium oxide (TSC) and MTA Plus (MTAP; Avalon Biomed Inc, Bradenton, FL) in rat subcutaneous tissues. METHODS: Polyethylene tubes were filled with TSC (n = 20) and MTAP (n = 20) and implanted in the dorsal subcutaneous tissues of 32 rats. Empty tubes were used as the control (control group [CG], n = 20). After 7, 15, 30, and 60 days, the tubes with connective tissue were removed, and the inflammatory cells and immunolabeled cells for interleukin 6 (IL-6) were counted. Data were statistically analyzed using analysis of variance and the Tukey test (P ≤ .05). RESULTS: An increased number of inflammatory and immunolabeled cells for IL-6 were observed at 7 days. The number of inflammatory cells was higher for TSC and MTAP than the CG (P < .001) at 7 days; after 30 and 60 days, no significant differences were observed among the TSC, MTAP, and CG (P = .955). The number of immunolabeled cells for IL-6 was similar for TSC, MTAP, and CG at all evaluated periods. A gradual and significant decrease was observed in the number of inflammatory cells and IL-6-immunopositive cells. At 60 days, the capsules adjacent to TSC and MTAP exhibited fibroblasts and bundles of collagen fibers. CONCLUSIONS: TSC and MTAP caused a similar subcutaneous reaction in rats, suggesting that they are biocompatible and present similar immune responses.

Influence of different sized files on the accuracy of two electronic apex locators.

The aim of this study was to evaluate the impact of different file sizes on the accuracy of two electronic apex locators (EALs). Thirty extracted human single-rooted permanent mandibular incisors were used. A #10 K-file was inserted in the root canal until its end could be observed (using a light microscope) through the apical foramen. One millimetre was subtracted to establish working length (WL). Electronic readings were performed using MiniApex Locator or Root ZX II, from #10 K-file to #130 K-file. Statistical analysis was performed by two-way anova and Tukey test (P ≤ 0.05). From #60 to #130 K-file, observed differences were noted between the values obtained with both EALs and WL (P ≤ 0.05). The MiniApex Locator showed increased means when measurements were made with #50 to #70 and with #120 (P = 0.008) and #130 (P = 0.005) K-files. File sizes influenced the accuracy of EALs - the greater the instrumentation size, the higher mean differences compared to WL.

The galactose-induced decrease in phosphate levels leads to toxicity in yeast models of galactosemia.

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations in the GALT gene. A number of evidences indicate that the galactose-1-phosphate accumulation observed in patient cells is a cause of toxicity in this disease. Nevertheless, the consequent molecular events caused by the galactose-1-phosphate accumulation remain elusive. Here we show that intracellular inorganic phosphate levels decreased when yeast models of classic galactosemia were exposed to galactose. The decrease in phosphate levels is probably due to the trapping of phosphate in the accumulated galactose-1-phosphate since the deletion of the galactokinase encoding gene GAL1 suppressed this phenotype. Galactose-induced phosphate depletion caused an increase in glycogen content, an expected result since glycogen breakdown by the enzyme glycogen phosphorylase is dependent on inorganic phosphate. Accordingly, an increase in intracellular phosphate levels suppressed the galactose effect on glycogen content and conferred galactose tolerance to yeast models of galactosemia. These results support the hypothesis that the galactose-induced decrease in phosphate levels leads to toxicity in galactosemia and opens new possibilities for the development of better treatments for this disease.

Niobium pentoxide as radiopacifying agent of calcium silicate-based material: evaluation of physicochemical and biological properties.

OBJECTIVES: The physicochemical properties and the tissue reaction promoted by microparticulated or nanoparticulated niobium pentoxide (Nb2O5) added to calcium silicate-based cement (CS), compared to MTA-Angelus™, were evaluated. MATERIALS AND METHODS: Materials were submitted to the tests of radiopacity, setting time, pH, and calcium ion release. Polyethylene tubes filled with the materials were implanted into rats subcutaneously. After 7, 15, 30, and 60 days, the specimens were fixed and embedded in paraffin. Hematoxylin & eosin (H&E)-stained sections were used to compute the number of inflammatory cells (IC). Interleukin-6 (IL-6) detection was performed, and the number of immunolabeled cells was obtained; von Kossa method was also carried out. Data were subjected to ANOVA and Tukey test (p ≤ 0.05). RESULTS: Nb2O5micro and Nb2O5nano provided to the CS radiopacity values (3.52 and 3.75 mm Al, respectively) superior to the minimum recommended. Groups containing Nb2O5 presented initial setting time significantly superior than mineral trioxide aggregate (MTA). All materials presented an alkaline pH and released calcium ions. The number of IC and IL-6 immunolabeled cells in the CS + Nb2O5 groups was significantly reduced in comparison to MTA in all periods. von Kossa-positive structures were observed adjacent to implanted materials in all periods. CONCLUSIONS: The addition of Nb2O5 to the CS resulted in a material biocompatible and with adequate characteristics regarding radiopacity and final setting time and provides an alkaline pH to the environment. Furthermore, the particle size did not significantly affect the physicochemical and biological properties of the calcium silicate-based cement. CLINICAL RELEVANCE: Niobium pentoxide can be used as radiopacifier for the development of calcium silicate-based materials.

Microparticulated and nanoparticulated zirconium oxide added to calcium silicate cement: Evaluation of physicochemical and biological properties.

The physicochemical and biological properties of calcium silicate-based cement (CS) associated to microparticulated (micro) or nanoparticulated (nano) zirconium oxide (ZrO2 ) were compared with CS and bismuth oxide (BO) with CS. The pH, release of calcium ions, radiopacity, setting time, and compression strength of the materials were evaluated. The tissue reaction promoted by these materials in the subcutaneous was also investigated by morphological, immunohistochemical, and quantitative analyses. For this purpose, polyethylene tubes filled with materials were implanted into rat subcutaneous. After 7, 15, 30, and 60 days, the tubes surrounded by capsules were fixed and embedded in paraffin. In the H&E-stained sections, the number of inflammatory cells (ICs) in the capsule was obtained. Moreover, detection of interleukin-6 (IL-6) by immunohistochemistry and number of IL-6 immunolabeled cells were carried out. von Kossa method was also performed. The differences among the groups were subjected to Tukey test (p ≤ 0.05). The solutions containing the materials presented an alkaline pH and released calcium ions. The addition of radiopacifiers increased setting time and radiopacity of CS. A higher compressive strength in the CS + ZrO2 (micro and nano) was found compared with CS + BO. The number of IC and IL-6 positive cells in the materials with ZrO2 was significantly reduced in comparison with CS + BO. von Kossa-positive structures were observed adjacent to implanted materials. The ZrO2 associated to the CS provides satisfactory physicochemical properties and better biological response than BO. Thus, ZrO2 may be a good alternative for use as radiopacifying agent in substitution to BO.

Comparison of face-to-face interview and telephone interview administration of COPD assessment test: a randomized study.

OBJECTIVE: To assess the measurement properties of the COPD assessment test (CAT) in a randomized trial comparing a face-to-face interview (FFI) with a telephone interview (TI). METHODS: A randomized study was conducted at two teaching hospitals in Fortaleza, Brazil. Patients were randomly assigned to answer the CAT questionnaire either in a FFI or by TI. The two groups were assessed for internal consistency reliability, cross-sectional validity and test-retest reliability. All patients performed spirometry and answered the modified medical research council dyspnea scale and the St. George's respiratory questionnaire (SGRQ). RESULTS: The total scores of the CAT questionnaire were similar for face-to-face and TI groups, 20.71 (95 % CI 18-23.4) versus 20.81 (95 % CI 19.31-21.7), respectively. For both mode of administration, we found good internal consistency reliability, the Cronbach's alpha ranged from 0.74 (95 % CI 0.61-0.84) to 0.89 (95 % CI 0.84-0.93) for the TI and FFI, respectively. In general, moderate-to-high correlations of CAT with SGRQ were observed, independent of the administration format. For the test-retest reliability, the intraclass correlation coefficients were very similar for both FFI and TI group 0.96 (95 % CI 0.93-0.97) versus 0.98 (95 % CI 0.96-0.98), respectively. CONCLUSION: This study demonstrated that the CAT questionnaire administration either in a FFI or by TI presents moderate-to-high measurement properties. This provides support for the use of both modes of questionnaire administration.

Preoperative pulmonary rehabilitation versus chest physical therapy in patients undergoing lung cancer resection: a pilot randomized controlled trial.

OBJECTIVE: To evaluate the effect of 4 weeks of pulmonary rehabilitation (PR) versus chest physical therapy (CPT) on the preoperative functional capacity and postoperative respiratory morbidity of patients undergoing lung cancer resection. DESIGN: Randomized single-blinded study. SETTING: A teaching hospital. PARTICIPANTS: Patients undergoing lung cancer resection (N=24). INTERVENTIONS: Patients were randomly assigned to receive PR (strength and endurance training) versus CPT (breathing exercises for lung expansion). Both groups received educational classes. MAIN OUTCOME MEASURES: Functional parameters assessed before and after 4 weeks of PR or CPT (phase 1), and pulmonary complications assessed after lung cancer resection (phase 2). RESULTS: Twelve patients were randomly assigned to the PR arm and 12 to the CPT arm. Three patients in the CPT arm were not submitted to lung resection because of inoperable cancer. During phase 1 evaluation, most functional parameters in the PR group improved from baseline to 1 month: forced vital capacity (FVC) (1.47L [1.27-2.33L] vs 1.71L [1.65-2.80L], respectively; P=.02); percentage of predicted FVC (FVC%; 62.5% [49%-71%] vs 76% [65%-79.7%], respectively; P<.05); 6-minute walk test (425.5±85.3m vs 475±86.5m, respectively; P<.05); maximal inspiratory pressure (90±45.9cmH(2)O vs 117.5±36.5cmH(2)O, respectively; P<.05); and maximal expiratory pressure (79.7±17.1cmH(2)O vs 92.9±21.4cmH(2)O, respectively; P<.05). During phase 2 evaluation, the PR group had a lower incidence of postoperative respiratory morbidity (P=.01), a shorter length of postoperative stay (12.2±3.6d vs 7.8±4.8d, respectively; P=.04), and required a chest tube for fewer days (7.4±2.6d vs 4.5±2.9d, respectively; P=.03) compared with the CPT arm. CONCLUSIONS: These findings suggest that 4 weeks of PR before lung cancer resection improves preoperative functional capacity and decreases the postoperative respiratory morbidity.

Tooth embedding medium influences the accuracy of electronic apex locator.

This study evaluated the influence of tooth embedding media on the accuracy ofan electronic apex locator. The root canal length of 20 human mandibular canines was measured by inserting a 15 K-file into the root canal up to the apical foramen. The distance was measured with a digital caliper. The embedding media evaluated were alginate, saline, floral foam or gauze soaked in saline. Electronic root canal length measurement was performed with Root ZX II. Data were analysed using ANOVA for repeated measurements and Tukey test, at a significance level of 5%. There was no difference between the actual root canal length measurement and the electronic reading recorded with alginate medium. The readings obtained with the other media differed from the actual root canal length measurements. Alginate provided greater accuracy in electronic root canal length determination by Root ZX II than saline, floral foam and gauze.

Compressive Strength and Setting Time of MTA and Portland Cement Associated with Different Radiopacifying Agents.

Objective. The aim of this study was to evaluate the compressive strength and setting time of MTA and Portland cement (PC) associated with bismuth oxide (BO), zirconium oxide (ZO), calcium tungstate (CT), and strontium carbonate (SC). Methods. For the compressive strength test, specimens were evaluated in an EMIC DL 2000 apparatus at 0.5 mm/min speed. For evaluation of setting time, each material was analyzed using Gilmore-type needles. The statistical analysis was performed with ANOVA and the Tukey tests, at 5% significance. Results. After 24 hours, the highest values were found for PC and PC + ZO. At 21 days, PC + BO showed the lowest compressive strength among all the groups. The initial setting time was greater for PC. The final setting time was greater for PC and PC + CT, and MTA had the lowest among the evaluated materials (P < 0.05). Conclusion. The results showed that all radiopacifying agents tested may potentially be used in association with PC to replace BO.

Sealing ability of retrograde obturation materials containing calcium hydroxide or MTA.

Newly available materials for retrograde obturation should have their sealing properties evaluated. The goal of this study was to evaluate the sealing ability of Endo CPM sealer, an MTA-based endodontic cement. Single-rooted extracted human teeth were endodontically treated. After apical sectioning, retrograde cavities were prepared. Teeth were divided into five experimental groups (n=12), in which the following materials were used: Sealer 26 (S26), white Mineral Trioxide Aggregate (MTA), Endo CPM Sealer (CPM1), Endo CPM Sealer in thicker consistency (CPM 2), and zinc oxide and eugenol cement (ZOE), and two control groups (n=3). After retrograde obturation, the teeth were immersed in 0.2% rhodamine B dye for 48 hours in a vacuum chamber Marginal leakage data were subjected to ANOVA and Tukey tests at 5% significance level. S26 presented greater sealing ability (p<0.05) than ZOE, MTA, CPM1, and CPM2, all of which had similar results (p>0.05). We concluded that Sealer 26 has the greatest sealing ability. Endo CPM Sealer, with sealing ability similar to MTA, could be used as a retrograde obturation material.