Microtensile Bond Strength of Composite Resin Following the Use of Bromelain and Papain as Deproteinizing Agents on Etched Dentin: An In Vitro Study.

AIM AND OBJECTIVES: The aim of this study was to evaluate and compare the deproteinizing effect of sodium hypochlorite, bromelain, and papain on microtensile bond strength of composite resin to etched dentin. MATERIALS AND METHODS: Eighty freshly extracted permanent molars were wet grounded into a flat surface using a diamond disk to expose the superficial dentinal surface. Teeth were etched with 37% phosphoric acid for 15 seconds and rinsed with water and blot dried. Teeth were divided into four groups (n = 20) based on the method of dentin deproteinization. Group I: only etching; group II: deproteinized with 5.25% sodium hypochlorite for 1 minute; group III: deproteinized with 8% bromelain enzyme for 1 minute; and group IV: deproteinized with 8% papain enzyme for 1 minute. All the samples were washed off with distilled water to remove deproteinizing agents. Sample surfaces were blot dried and bonding of the dentin surface was performed and restored with light cure bulk fill composite. Samples were stored in distilled water (37°C/24 hours) and thermocycled. Then, the teeth were longitudinally sectioned and individually fixed to a sectioning block using acrylic resin. The block was mounted on hard tissue microtome and sectioned to get one to three slabs of 1 mm thick sections. The beam was then attached to a custom-made jig using screws subjected to the Instron universal testing machine. A tensile load was applied at a crosshead speed of 0.5 mm/minute until the beam fractured. RESULTS: Higher mean bond strength was recorded in group IV followed by group III, group II, and group I, respectively. Group III presented a statistically significant highest mean score compared to other study groups with group I and group II (p < 0.001), followed by group IV having significantly higher mean score compared to group I and group II (p < 0.001) and finally a significant difference was observed between group II and group I (p < 0.001). However, the mean microtensile bond strength score did not differ significantly between group III and group IV (p = 0.20). CONCLUSION: Within the limitations of this present in vitro study, the following conclusions were drawn. The microtensile bond strength of dentine tested in various deproteinizing agents is as follows: 8% bromelain > 8% papain > 5.25% NaOCl > control group. Naturally occurring deproteinizing agents, such as bromelain and papain, used in this study have resulted in greater bond strength values when compared to that of traditionally used chemical agent such as NaOCl. HOW TO CITE THIS ARTICLE: Khatib MS, Devarasanahalli SV, Aswathanarayana RM, et al. Microtensile Bond Strength of Composite Resin Following the Use of Bromelain and Papain as Deproteinizing Agents on Etched Dentin: An In Vitro Study. Int J Clin Pediatr Dent 2020;13(1):43-47.

Effect of 5% Chlorine Dioxide Irrigant on Micro Push Out Bond Strength of Resin Sealer to Radicular Dentin: An In Vitro Study.

INTRODUCTION: Chlorine dioxide (ClO2) has been recently investigated as a possible root canal irrigant due to its broad spectrum of antimicrobial action, tissue dissolution and smear layer removal properties. Literature is scarce on the effect of chlorine dioxide irrigation on the resin sealer dentin bond strength. AIM: To compare 5% chlorine dioxide (ClO2) with or without Ethylene Diamine Tetra Acetic acid (EDTA) with 3% Sodium hypochlorite (NaOCl) and EDTA combination as endodontic irrigants on the adhesion of AH Plus sealer to radicular dentin using micro- Push out Bond Strength (µPBS) test. MATERIALS AND METHODS: Forty freshly extracted central incisors were decoronated and randomly divided into four groups based on the different irrigation regimes followed during irrigation: Group I - 3% NaOCl + 17% EDTA, Group II - 5% ClO2 + 17% EDTA, Group III - 5% ClO2 and Group IV - Saline, and canal enlarged till Protaper F3. All the samples were obturated with F3 gutta-percha cones using AH Plus sealer and sectioned perpendicular to long axis to obtain 1mm thick slices from the middle and coronal portions for µPBS measurement in universal testing machine followed by assessment of failure pattern under stereomicroscope. Data was analysed using One-way analysis of variance (ANOVA), Bonferroni and t-test. RESULTS: Bond strength values were in the following order: Group I>Group II>Group III>Group IV, with no statistically significant difference amongst experimental groups on intergroup comparison, except with saline. The µPBS values were more in coronal third than middle third in all specimens, with no statistical significant difference. Mode of failure showed mixed patterns in all experimental groups except saline. CONCLUSION: In the present study, the bond strength values of ClO2 were comparable with conventional NaOCl and EDTA combination and hence, ClO2 can be considered as an effective alternative endodontic irrigant.

A moderate increase in dietary zinc reduces DNA strand breaks in leukocytes and alters plasma proteins without changing plasma zinc concentrations.

BACKGROUND: Food fortification has been recommended to improve a population's micronutrient status. Biofortification techniques modestly elevate the zinc content of cereals, but few studies have reported a positive impact on functional indicators of zinc status. OBJECTIVE: We determined the impact of a modest increase in dietary zinc that was similar to that provided by biofortification programs on whole-body and cellular indicators of zinc status. DESIGN: Eighteen men participated in a 6-wk controlled consumption study of a low-zinc, rice-based diet. The diet contained 6 mg Zn/d for 2 wk and was followed by 10 mg Zn/d for 4 wk. To reduce zinc absorption, phytate was added to the diet during the initial period. Indicators of zinc homeostasis, including total absorbed zinc (TAZ), the exchangeable zinc pool (EZP), plasma and cellular zinc concentrations, zinc transporter gene expression, and other metabolic indicators (i.e., DNA damage, inflammation, and oxidative stress), were measured before and after each dietary-zinc period. RESULTS: TAZ increased with increased dietary zinc, but plasma zinc concentrations and EZP size were unchanged. Erythrocyte and leukocyte zinc concentrations and zinc transporter expressions were not altered. However, leukocyte DNA strand breaks decreased with increased dietary zinc, and the level of proteins involved in DNA repair and antioxidant and immune functions were restored after the dietary-zinc increase. CONCLUSIONS: A moderate 4-mg/d increase in dietary zinc, similar to that which would be expected from zinc-biofortified crops, improves zinc absorption but does not alter plasma zinc. The repair of DNA strand breaks improves, as do serum protein concentrations that are associated with the DNA repair process. This trial was registered at clinicaltrials.gov as NCT02861352.

Acute changes in cellular zinc alters zinc uptake rates prior to zinc transporter gene expression in Jurkat cells.

A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.

A multicomponent nutrient bar promotes weight loss and improves dyslipidemia and insulin resistance in the overweight/obese: chronic inflammation blunts these improvements.

This study determined if twice-daily consumption of a nutrient-dense bar intended to fill gaps in Western diets, without other dietary/lifestyle requirements, favorably shifted metabolic/anthropometric indicators of dysregulation in a healthy direction. Three 8-wk clinical trials in 43 healthy lean and overweight/obese (OW/OB) adults, who served as their own controls, were pooled for analysis. In less inflamed OW/OB [high-sensitivity C-reactive protein (hsCRP) <1.5], statistically significant decreases occurred in weight (-1.1 ± 0.5 kg), waist circumference (-3.1 ± 1.4 cm), diastolic blood pressure (-4.1 ± 1.6 mmHg), heart rate [HR; -4.0 ± 1.7 beats per minute (bpm)], triglycerides (-72 ± 38.2 mg/dl), insulin resistance (homeostatic model of insulin resistance) (-0.72 ± 0.3), and insulin (-2.8 ± 1.3 mU/L); an increase in HDL-2b (+303 ± 116 nM) and realignment of LDL lipid subfractions toward a less atherogenic profile [decreased small LDL IIIb (-44 ± 23.5 nM), LDL IIIa (-99 ± 43.7 nM), and increased large LDL I (+66 ± 28.0 nM)]. In the more inflamed OW/OB (hsCRP >1.5), inflammation was reduced at 2 wk (-0.66 mg/L), and HR at 8 wk (-3.4 ± 1.3 bpm). The large HDL subfraction (10.5-14.5 nm) increased at 8 wk (+346 ± 126 nM). Metabolic improvements were also observed in lean participants. Thus, favorable changes in measures of cardiovascular health, insulin resistance, inflammation, and obesity were initiated within 8 wk in the OW/OB by replacing deficiencies in Western diets without requiring other dietary or lifestyle modifications; chronic inflammation blunted most improvements.

A nutrient-dense, high-fiber, fruit-based supplement bar increases HDL cholesterol, particularly large HDL, lowers homocysteine, and raises glutathione in a 2-wk trial.

Dietary intake modulates disease risk, but little is known how components within food mixtures affect pathophysiology. A low-calorie, high-fiber, fruit-based nutrient-dense bar of defined composition (e.g., vitamins and minerals, fruit polyphenolics, ß-glucan, docosahexaenoic acid) appropriate for deconstruction and mechanistic studies is described and evaluated in a pilot trial. The bar was developed in collaboration with the U.S. Department of Agriculture. Changes in cardiovascular disease and diabetes risk biomarkers were measured after 2 wk twice-daily consumption of the bar, and compared against baseline controls in 25 healthy adults. Plasma HDL-cholesterol (HDL-c) increased 6.2% (P=0.001), due primarily to a 28% increase in large HDL (HDL-L; P<0.0001). Total plasma homocysteine (Hcy) decreased 19% (P=0.017), and glutathione (GSH) increased 20% (P=0.011). The changes in HDL and Hcy are in the direction associated with decreased risk of cardiovascular disease and cognitive decline; increased GSH reflects improved antioxidant defense. Changes in biomarkers linked to insulin resistance and inflammation were not observed. A defined food-based supplement can, within 2 wk, positively impact metabolic biomarkers linked to disease risk. These results lay the groundwork for mechanistic/deconstruction experiments to identify critical bar components and putative synergistic combinations responsible for observed effects.

Identification of age-specific Nrf2 binding to a novel antioxidant response element locus in the Gclc promoter: a compensatory means for the loss of glutathione synthetic capacity in the aging rat liver?

NFE2-related factor 2 (Nrf2) transcriptionally governs the cellular response to harmful electrophiles, xenobiotics, and reactive oxygen species. Its nuclear levels decline with age (Suh et al., 2004a), which in part explains the age-related loss of phase II detoxification. However, little work has yet characterized how age affects Nrf2 DNA binding or the role that alterations to the Nrf2 transcriptional apparatus plays in modulating Nrf2-mediated gene expression. In this study, we used immunoprecipitation assays to show that Nrf2 bound to the active antioxidant response element (ARE) of the catalytic subunit of glutamate cysteine ligase (GCLC) is significantly lower in hepatic chromatin from aged vs. young rats. Moreover, the activity at this ARE locus is diminished during aging because of the presence of Bach1 and the absence of CREB-binding protein (CBP), a transcriptional repressor and co-activator, respectively. Further analysis reveals that Nrf2 occupies an alternate ARE site located -2.2 kb downstream from the normally active ARE binding site in livers of old rats, indicating an age-specific adaptation to maintain gene expression. Our results, thus, show that the conversion of Nrf2 binding from an active ARE to an alternative ARE element is not adequate to maintain basal expression of hepatic Gclc in old rats, which provides a potential mechanism for the age-related loss of glutathione synthetic and other phase II enzymes.

Transcriptional regulation of rat gamma-glutamate cysteine ligase catalytic subunit gene is mediated through a distal antioxidant response element.

Despite it being a quintessential Phase II detoxification gene, the transcriptional regulation of the rat gamma-glutamate cysteine ligase catalytic subunit (GCLC) is controversial. Computer-based sequence analysis identified three putative antioxidant response elements (AREs) at positions -889 to -865 (ARE1), -3170 to -3146 (ARE2) and -3901 to -3877 (ARE3) in the 5'-flanking region of the transcriptional start site. Transfections of individual ARE-luciferase reporter gene constructs into H4IIE cells, a rat hepatoma cell line, identified ARE3 as the functional promoter. Chromatin immunoprecipitation assays using primary rat hepatocytes showed that the transcription factor Nrf2, which is known to regulate ARE-mediated genes, is associated with ARE3. Co-transfection of H4IIE cells with luciferase reporter plasmids containing Gclc ARE3 and an Nrf2 expression plasmid resulted in a 3-fold activation of ARE3-mediated transcription relative to controls. "Loss-of-function" analysis for Nrf2 by small interfering RNA (siRNA) revealed that ARE3-mediated expression was significantly impaired while site-directed mutagenesis of the ARE3-luciferase reporter abolished Nrf2-mediated induction. Treatment with two known Nrf2 inducers, R-(alpha)-lipoic acid and anetholedithiolethione, showed that the inducible expression of the GCLC gene was also regulated by the ARE3 element. Taken together, these results show that Nrf2 regulates the constitutive expression of rat Gclc through a distal ARE present in its 5'-flanking region. This is the first report showing that rat Gclc is under the transcriptional control of the Nrf2-ARE pathway on a constitutive basis.

A rat primary hepatocyte culture model for aging studies.

The purpose of this protocol is to establish a primary hepatocyte culture system as a suitable model to examine age-related changes in Phase II detoxication gene expression. Hepatocytes are isolated using a two-step collagenase perfusion technique from young (3 to 6 months) and old (24 to 28 months) rats and placed in primary culture using collagen (Type I)-coated plates as the extracellular matrix. A supplemented William's E Medium is used as the medium. This culture system maintains hepatocyte viability from both young and old rats for ∼60 hr, as measured by lactate dehydrogenase activity, while also maintaining their respective phenotypes relative to Phase II detoxification. We thus conclude that a collagen-based cell culture system is suitable to study age-associated deficits in Nrf2/ARE-mediated Phase II gene regulation provided that experiments can be conducted within 60 hr after cell isolation.

Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acid.

Glutathione (GSH) significantly declines in the aging rat liver. Because GSH levels are partly a reflection of its synthetic capacity, we measured the levels and activity of gamma-glutamylcysteine ligase (GCL), the rate-controlling enzyme in GSH synthesis. With age, both the catalytic (GCLC) and modulatory (GCLM) subunits of GCL decreased by 47% and 52%, respectively (P < 0.005). Concomitant with lower subunit levels, GCL activity also declined by 53% (P < 0.05). Because nuclear factor erythroid2-related factor 2 (Nrf2) governs basal and inducible GCLC and GCLM expression by means of the antioxidant response element (ARE), we hypothesized that aging results in dysregulation of Nrf2-mediated GCL expression. We observed an approximately 50% age-related loss in total (P < 0.001) and nuclear (P < 0.0001) Nrf2 levels, which suggests attenuation in Nrf2-dependent gene transcription. By using gel-shift and supershift assays, a marked reduction in Nrf2/ARE binding in old vs. young rats was noted. To determine whether the constitutive loss of Nrf2 transcriptional activity also affects the inducible nature of Nrf2 nuclear translocation, old rats were treated with (R)-alpha-lipoic acid (LA; 40 mg/kg i.p. up to 48 h), a disulfide compound shown to induce Nrf2 activation in vitro and improve GSH levels in vivo. LA administration increased nuclear Nrf2 levels in old rats after 12 h. LA also induced Nrf2 binding to the ARE, and, consequently, higher GCLC levels and GCL activity were observed 24 h after LA injection. Thus, the age-related loss in GSH synthesis may be caused by dysregulation of ARE-mediated gene expression, but chemoprotective agents, like LA, can attenuate this loss.

Psychiatric comorbidity in first episode psychosis: the Early Psychosis Intervention Program (EPIP) experience.

OBJECTIVE: To determine the prevalence rates of psychiatric comorbidity in a hospitalized Asian patient group with first episode psychosis and examine its clinical correlates. METHOD: Seventy-nine consecutively admitted patients with first episode psychosis were assessed using the Structured Clinical Interview for DSM-IV-axis I disorders (patient edition), Positive and Negative Symptom Scale (PANSS), Scale to assess Unawareness of Mental Disorders (SUMD) and Global Assessment of Functioning (GAF) scales. RESULTS: Psychiatric comorbidity was present in 36.7% (n = 29) of the patients. Patients with psychiatric comorbidity were younger (P < 0.05), had an earlier onset of illness (P < 0.05) and better insight on social consequences and flat affect items (P < 0.05) on the SUMD. No significant differences were found between the two groups with and without psychiatric comorbidity in gender, ethnicity, marital status, length of education, employment status, living arrangements, duration of hospitalization and untreated psychosis as well as total PANSS and GAF scores. CONCLUSION: Psychiatric comorbidity is common thus calling for a greater awareness in clinicians of these conditions, which are often under-recognized, under-diagnosed and untreated.