Effect of novel biomodification strategies on bonding to pulp chamber dentin - An In Vitro study.

Context: Adhesion to dentin remains a tough challenge due to its heterogeneous composition, complex histologic structure and high tubular content, warranting the need to investigate methods to improve the bond strength of the commonly used access restorative materials to pulp chamber dentin. Aims: To evaluate the effect of dentin biomodification using 6.5% grape seed extract and a 980 nm diode LASER on the shear bond strength of resin-based bonded restoration to pulp chamber dentin. Methods and Materials: Access cavities were prepared in 42 extracted human maxillary premolars, which were then sectioned in a buccolingual direction. The samples were serially immersed in 5.25% NaOCl for 40 minutes and 17% EDTA for 3 minutes and allocated into three groups: the control group, the group pre-treated with 6.5% grape seed extract (GSE) and the group pre-treated with a 980 nm diode light amplification by stimulated emission of radiation (LASER). All the samples were restored with resin composites and subjected to shear bond strength testing using a universal testing machine. Statistical Analysis Used: Statistical analysis was done using SPSS version 23 software. Results: The mean shear bond strength was highest in the group pre-treated with GSE, followed by that pre-treated with diode LASER and finally in the control group. Conclusions: Dentin biomodification using both chemical and physical agents such as grape seed extract and diode LASER was shown to improve the shear bond strength of resin composite endodontic access restorations to the pulp chamber dentin.

Association of CNVs with methylation variation.

Germline copy number variants (CNVs) and single-nucleotide polymorphisms (SNPs) form the basis of inter-individual genetic variation. Although the phenotypic effects of SNPs have been extensively investigated, the effects of CNVs is relatively less understood. To better characterize mechanisms by which CNVs affect cellular phenotype, we tested their association with variable CpG methylation in a genome-wide manner. Using paired CNV and methylation data from the 1000 genomes and HapMap projects, we identified genome-wide associations by methylation quantitative trait locus (mQTL) analysis. We found individual CNVs being associated with methylation of multiple CpGs and vice versa. CNV-associated methylation changes were correlated with gene expression. CNV-mQTLs were enriched for regulatory regions, transcription factor-binding sites (TFBSs), and were involved in long-range physical interactions with associated CpGs. Some CNV-mQTLs were associated with methylation of imprinted genes. Several CNV-mQTLs and/or associated genes were among those previously reported by genome-wide association studies (GWASs). We demonstrate that germline CNVs in the genome are associated with CpG methylation. Our findings suggest that structural variation together with methylation may affect cellular phenotype.

Neuron Activity Dependent Redox Compartmentation Revealed with a Second Generation Red-Shifted Ratiometric Sensor.

Oxidative stress is a hallmark of several aging and trauma related neurological disorders, but the precise details of how altered neuronal activity elicits subcellular redox changes have remained difficult to resolve. Current redox sensitive dyes and fluorescent proteins can quantify spatially distinct changes in reactive oxygen species levels, but multicolor probes are needed to accurately analyze compartment-specific redox dynamics in single cells that can be masked by population averaging. We previously engineered genetically encoded red-shifted redox-sensitive fluorescent protein sensors using a Förster resonance energy transfer relay strategy. Here, we developed a second-generation excitation ratiometric sensor called rogRFP2 with improved red emission for quantitative live-cell imaging. Using this sensor to measure activity-dependent redox changes in individual cultured neurons, we observed an anticorrelation in which mitochondrial oxidation was accompanied by a concurrent reduction in the cytosol. This behavior was dependent on the activity of Complex I of the mitochondrial electron transport chain and could be modulated by the presence of cocultured astrocytes. We also demonstrated that the red fluorescent rogRFP2 facilitates ratiometric one- and two-photon redox imaging in rat brain slices and Drosophila retinas. Overall, the proof-of-concept studies reported here demonstrate that this new rogRFP2 redox sensor can be a powerful tool for understanding redox biology both in vitro and in vivo across model organisms.

Shh-Mediated Increase in ß-Catenin Levels Maintains Cerebellar Granule Neuron Progenitors in Proliferation.

Cerebellar granule neuron progenitors (CGNPs) give rise to the cerebellar granule neurons in the developing cerebellum. Generation of large number of these neurons is made possible by the high proliferation rate of CGNPs in the external granule layer (EGL) in the dorsal cerebellum. Here, we show that upregulation of ß-catenin can maintain murine CGNPs in a state of proliferation. Further, we show that ß-catenin mRNA and protein levels can be regulated by the mitogen Sonic hedgehog (Shh). Shh signaling led to an increase in the level of the transcription factor N-myc. N-myc was found to bind the ß-catenin promoter, and the increase in ß-catenin mRNA and protein levels could be prevented by blocking N-myc upregulation downstream of Shh signaling. Furthermore, blocking Wingless-type MMTV integration site (Wnt) signaling by Wnt signaling pathway inhibitor Dickkopf 1 (Dkk-1) in the presence of Shh did not prevent the upregulation of ß-catenin. We propose that in culture, Shh signaling regulates ß-catenin expression through N-myc and results in increased CGNP proliferation.

Imaging extracellular ATP with a genetically-encoded, ratiometric fluorescent sensor.

Extracellular adenosine triphosphate (ATP) is a key purinergic signal that mediates cell-to-cell communication both within and between organ systems. We address the need for a robust and minimally invasive approach to measuring extracellular ATP by re-engineering the ATeam ATP sensor to be expressed on the cell surface. Using this approach, we image real-time changes in extracellular ATP levels with a sensor that is fully genetically-encoded and does not require an exogenous substrate. In addition, the sensor is ratiometric to allow for reliable quantitation of extracellular ATP fluxes. Using live-cell microscopy, we characterize sensor performance when expressed on cultured Neuro2A cells, and we measure both stimulated release of ATP and its clearance by ectonucleotidases. Thus, this proof-of-principle demonstrates a first-generation sensor to report extracellular ATP dynamics that may be useful for studying purinergic signaling in living specimens.

Survival of Del17p CLL Depends on Genomic Complexity and Somatic Mutation.

PURPOSE: Chronic lymphocytic leukemia (CLL) with 17p deletion typically progresses quickly and is refractory to most conventional therapies. However, some del(17p) patients do not progress for years, suggesting that del(17p) is not the only driving event in CLL progression. We hypothesize that other concomitant genetic abnormalities underlie the clinical heterogeneity of del(17p) CLL. EXPERIMENTAL DESIGN: We profiled the somatic mutations and copy number alterations (CNA) in a large group of del(17p) CLLs as well as wild-type CLL and analyzed the genetic basis of their clinical heterogeneity. RESULTS: We found that increased somatic mutation number associates with poor overall survival independent of 17p deletion (P = 0.003). TP53 mutation was present in 81% of del(17p) CLL, mostly clonal (82%), and clonal mutations with del(17p) exhibit shorter overall survival than subclonal mutations with del(17p) (P = 0.019). Del(17p) CLL has a unique driver mutation profile, including NOTCH1 (15%), RPS15 (12%), DDX3X (8%), and GPS2 (6%). We found that about half of del(17p) CLL cases have recurrent deletions at 3p, 4p, or 9p and that any of these deletions significantly predicts shorter overall survival. In addition, the number of CNAs, but not somatic mutations, predicts shorter time to treatment among patients untreated at sampling. Indolent del(17p) CLLs were characterized by absent or subclonal TP53 mutation and few CNAs, with no difference in somatic mutation number. CONCLUSIONS: We conclude that del(17p) has a unique genomic profile and that clonal TP53 mutations, 3p, 4p, or 9p deletions, and genomic complexity are associated with shorter overall survival. Clin Cancer Res; 23(3); 735-45. ©2016 AACR.

Diabetic kidney disease patients on hemodialysis: a retrospective survival analysis across different socioeconomic groups.

BACKGROUND: Diabetic kidney disease is the leading cause of stage 5 chronic kidney disease (CKD) in India. Renal replacement therapy (RRT) is accessible to very few patients because of socioeconomic deprivation. We studied the effect of diabetes and socioeconomic status on the outcome of patients on maintenance hemodialysis (MHD). METHODS: We retrospectively analyzed the outcome of 897 patients (629 males/268 females; mean age ± standard deviation 48.69 ± 14.27 years) initiated on MHD from 2003 to 2009 at five dialysis centers in south India. There were 335 type 2 diabetic patients and 562 non-diabetic patients. Group 1 comprised the self-paying patients (518 patients) and Group 2 included the TANKER Foundation charity dialysis patients (379 patients). We compared the 5-year survival rates of Group 1 versus Group 2 and also those of diabetic versus non-diabetic patients, using the Kaplan-Meier survival estimator. RESULTS: Of the 897 patients, 166 patients survived, 350 died, 234 were lost to follow-up, 137 had renal transplantation and 10 patients were transferred to peritoneal dialysis. The 5-year survival rates after censoring were 20.7 and 38.2% for diabetic and non-diabetic patients, respectively (P < 0.001). The survival rate of diabetic patients was significantly lower, compared with non-diabetic patients, in Group 2 (P < 0.001), but not significantly lower in Group 1 (P = 0.226). CONCLUSIONS: Diabetic patients have poor survival rates on MHD, especially those from poor socioeconomic groups. Due to scarce RRT facilities and poor survival rates of diabetic patients, prevention, early detection and management of diabetic CKD patients should be the way to go forward.