Assessment of the Effect of Complete Dentures on Respiratory Performance: A Spirometric Analysis.

BACKGROUND: Among the vital functions, respiration can be claimed to be one of the most important. A thorough understanding of the patient should be an important aspect a prosthodontist should dwell into so as to predispose patients to acknowledge the kind of prosthesis they require. Hence, the present study was conducted for assessing the effect of complete dentures on respiratory performance. MATERIALS AND METHODS: Fifty patients with the presence of complete edentulous arch and who had a history of complete denture usage for at least 5 years were enrolled. All the spirometric procedures were performed by trained technicians. A diagnostic spirometer was employed for performing the spirometric test. Testing was carried out in the following steps: Stage 1: testing in the absence of denture, Stage 2: testing in the presence of both dentures, Stage 3: testing in the presence of maxillary denture only, and Stage 4: testing in the presence of mandibular dentures only. Forced vital capacity (FVC) value, peak expiratory flow (PEF) value, forced expiratory volume in 1 s (FEV1) value, and forced expiratory flow 25%-75% (FEF25-75) value were recorded with the spirometric test. Analysis of all the results was done by SPSS software. RESULTS: The spirometric value of FVC, PEF, FEV1, and FEF25-75 in the absence of both maxillary and mandibular dentures (Stage 1) was found to be 3.18, 5.83, 2.44, and 2.80, respectively. The spirometric value of FVC, PEF, FEV1, and FEF25-75 in the presence of both maxillary and mandibular dentures (Stage 2) was found to be 3.09, 5.67, 2.41, and 2.67, respectively. While analyzing statistically, it was seen that there was a significant decrease in the value of spirometric variables in the presence of dentures. CONCLUSION: Chronic denture wearer edentulous patients are subjected to the risk of development of spirometric alterations. Hence, these patients should be given timely instructions about the various respiratory exercise protocols.

PSMA-targeted bispecific Fab conjugates that engage T cells.

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity.

Wnt5a Drives an Invasive Phenotype in Human Glioblastoma Stem-like Cells.

Brain invasion by glioblastoma determines prognosis, recurrence, and lethality in patients, but no master factor coordinating the invasive properties of glioblastoma has been identified. Here we report evidence favoring such a role for the noncanonical WNT family member Wnt5a. We found the most invasive gliomas to be characterized by Wnt5a overexpression, which correlated with poor prognosis and also discriminated infiltrating mesenchymal glioblastoma from poorly motile proneural and classical glioblastoma. Indeed, Wnt5a overexpression associated with tumor-promoting stem-like characteristics (TPC) in defining the character of highly infiltrating mesenchymal glioblastoma cells (Wnt5aHigh). Inhibiting Wnt5a in mesenchymal glioblastoma TPC suppressed their infiltrating capability. Conversely, enforcing high levels of Wnt5a activated an infiltrative, mesenchymal-like program in classical glioblastoma TPC and Wnt5aLow mesenchymal TPC. In intracranial mouse xenograft models of glioblastoma, inhibiting Wnt5a activity blocked brain invasion and increased host survival. Overall, our results highlight Wnt5a as a master regulator of brain invasion, specifically TPC, and they provide a therapeutic rationale to target it in patients with glioblastoma. Cancer Res; 77(4); 996-1007. ©2016 AACR.

A comparative evaluation of static frictional resistance using various methods of ligation at different time intervals: an in vitro study.

Aim. To compare and evaluate the static frictional resistance offered by the four different types of ligation methods in both dry and wet conditions and at different durations when immersed in artificial saliva. Material and Methods. Alastik Easy to Tie modules, Super Slick Mini Stix elastomeric modules, Power "O" modules, and 0.009(″) Stainless Steel ligatures were used to compare the static friction using maxillary canine and premolar Preadjusted Edgewise brackets with 0.022(″) × 0.028(″) slot and 0.019(″) × 0.025(″) stainless steel wires. Results. The mean frictional resistance for Alastik modules was the lowest and that of Stainless Steel ligatures was found to be highest among the four groups compared and the difference among the four groups was statistically significant (P < 0.005). The mean static frictional resistance in all groups under dry conditions was lower than that under wet conditions. No statistical significant differences were found when the groups were compared at different time periods of immersion in artificial saliva. Conclusion. This study concludes that the Alastik modules showed the lowest mean static frictional forces compared to any other ligation method, though no significant difference was found for different time periods of immersion in the artificial saliva.

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines.

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.

Vaccinia virus expressing bone morphogenetic protein-4 in novel glioblastoma orthotopic models facilitates enhanced tumor regression and long-term survival.

BACKGROUND: Glioblastoma multiforme (GBM) is one of the most aggressive forms of cancer with a high rate of recurrence. We propose a novel oncolytic vaccinia virus (VACV)-based therapy using expression of the bone morphogenetic protein (BMP)-4 for treating GBM and preventing recurrence. METHODS: We have utilized clinically relevant, orthotopic xenograft models of GBM based on tumor-biopsy derived, primary cancer stem cell (CSC) lines. One of the cell lines, after being transduced with a cDNA encoding firefly luciferase, could be used for real time tumor imaging. A VACV that expresses BMP-4 was constructed and utilized for infecting several primary glioma cultures besides conventional serum-grown glioma cell lines. This virus was also delivered intracranially upon implantation of the GBM CSCs in mice to determine effects on tumor growth. RESULTS: We found that the VACV that overexpresses BMP-4 demonstrated heightened replication and cytotoxic activity in GBM CSC cultures with a broad spectrum of activity across several different patient-biopsy cultures. Intracranial inoculation of mice with this virus resulted in a tumor size equal to or below that at the time of injection. This resulted in survival of 100% of the treated mice up to 84 days post inoculation, significantly superior to that of a VACV lacking BMP-4 expression. When mice with a higher tumor burden were injected with the VACV lacking BMP-4, 80% of the mice showed tumor recurrence. In contrast, no recurrence was seen when mice were injected with the VACV expressing BMP-4, possibly due to induction of differentiation in the CSC population and subsequently serving as a better host for VACV infection and oncolysis. This lack of recurrence resulted in superior survival in the BMP-4 VACV treated group. CONCLUSIONS: Based on these findings we propose a novel VACV therapy for treating GBM, which would allow tumor specific production of drugs in the future in combination with BMPs which would simultaneously control tumor maintenance and facilitate CSC differentiation, respectively, thereby causing sustained tumor regression without recurrence.

Biotherapeutic approaches to target cancer stem cells.

With the cementing of the cancer stem cell (CSC) concept, cancer biology and cancer drug discovery have attained a new avenue to target cancer. Studying the hierarchy of tumor tissue organization and how to inhibit the cell that resides at the very top of this hierarchy has opened up a new branch of tumor biology and given the opportunity to develop novel cancer-targeting strategies. With the discovery of CSCs in majority of cancer indications there seems to be a universal applicability of the concept. However, the CSC field is still at an early fledgling state and a lot more needs to be done in terms of understanding their emergence, maintenance, role in metastasis and their function in shaping the tumor architecture. CSCs are considered to be responsible for tumor initiation, metastasis and resistance to conventional radio and chemotherapy. Therefore, different approaches to targeting these tumorigenic and rare cells are urgently needed in order to improve the efficacy of anti-cancer therapy. We outline here the cancer stem cell concept and its relevance as well as biotherapeutic approaches to CSC targeting, including oncolytic viruses, monoclonal antibodies, cytokines and cytotoxic T lymphocytes.

Functional role of solid tumor stem cells in disease etiology and susceptibility to therapeutic interventions.

Cancer stem cells (CSCs) are defined by their innate stem cell like properties and can be identified by specific markers that include antigens, molecules and signaling pathways. Like stem cells, CSC divide indefinitely giving rise to both more CSCs and differentiated cell progeny. CSCs can give rise to tumors that phenotypically resemble their origin, either morphologically or by expression of tissue specific genes. Tumors arise from a single cell, the CSC, but the cells that constitute the tumor are not identical to each other. Evidence of heterogeneous populations within a tumor has led to an investigation of the cellular hierarchy of cancers. This review gives an overview of cancer stem cells, from breast, cervical, lung, prostate, head and neck, glioblastoma, pancreatic and colorectal cancers and mechanisms implicated in tumor development and therapeutic interventions.

Biochemical characterization of recombinant hepatitis C virus nonstructural protein 4B: evidence for ATP/GTP hydrolysis and adenylate kinase activity.

While nonstructural protein 4B (NS4B) from hepatitis C virus (HCV) is absolutely required for viral propagation, a full understanding of the enzymatic properties of this protein is lacking. Previous studies suggest that NS4B is located at the endoplasmic reticulum and that the protein structure consists of four central transmembrane domains with the N- and C-termini located in the cytoplasm of the host cell. To characterize the enzymatic activity of NS4B, the full-length protein with a C-terminal His tag was expressed in Sf9 insect cells and stabilized with nonionic detergents during purification. Chemical cross-linking experiments using GTP-gamma-azidoanilide and ATP-gamma-azidoanilide and equilibrium binding analyses with GTPgammaS and ATPgammaS show that both GTP and ATP are bound by NS4B, with ATP displaying a higher affinity. Analyses of enzymatic reactions catalyzed by NS4B indicate that the terminal phosphate groups of ATP, GTP, and GDP are removed to produce ADP, GDP, and GMP, respectively. The k(cat) for hydrolysis of GTP by purified NS4B compared favorably with the k(cat) for hydrolysis of GTP by Ras-p21 in the absence of GTPase activating proteins (GAPs). In addition to the hydrolysis of NTP and NDP substrates, adenylate kinase activity was detected in purified preparations of NS4B with the reverse reaction 2ADP --> ATP + ADP, yielding a larger k(cat) compared to that of the forward reaction ATP + AMP --> 2ADP. These studies suggest that HCV NS4B possesses both adenylate kinase activity and nucleotide hydrolase activity. Mutation of amino acids in the Walker A and B motifs of NS4B resulted in decreased affinity for both GTPgammaS and ATPgammaS as well as decreased ATP hydrolysis and AK activity.

High-throughput screening of HCV RNA replication inhibitors by means of a reporter replicon system.

Efforts to find effective treatment for hepatitis C virus (HCV) have been hampered by the lack of a robust in vitro infectious tissue-culture system for this virus. A subgenomic replicon system was first developed in 1999 and has since been extensively optimized to accommodate the need for conveniently measuring HCV replication in vitro and widely adopted in HCV drug-discovery efforts. Here we describe the adaptation of a modified replicon system for a high-throughput screening (HTS) in anti-HCV drug discovery. In this system, the antiviral activity and cytotoxicity of any experimental compound are measured from a single well. This duplex measurement greatly increases the efficiency of the HTS while lowering the cost. The usefulness of this approach has been supported by the recent discovery of many new lead compounds from our HTS efforts in the past two years.

Development of intergenotypic chimeric replicons to determine the broad-spectrum antiviral activities of hepatitis C virus polymerase inhibitors.

To address the need for broad-spectrum antiviral activity characterization of hepatitis C virus (HCV) polymerase inhibitors, we created a panel of intergenotypic chimeric replicons containing nonstructural (NS) protein NS5B sequences from genotype 2b (GT2b), GT3a, GT4a, GT5a, and GT6a HCV isolates. Viral RNA extracted from non-GT1 HCV patient plasma was subjected to reverse transcription. The NS5B region was amplified by nested PCR and introduced into the corresponding region of the GT1b (Con-1) subgenomic reporter replicon by Splicing by Overlap Extension (SOEing) PCR. Stable cell lines were generated with replication-competent chimeras for in vitro antiviral activity determination of HCV nonnucleoside polymerase inhibitors (NNIs) that target different regions of the protein. Compounds that bind to the NNI2 (thiophene carboxylic acid) or NNI3 (benzothiadiazine) allosteric sites showed 8- to >1,280-fold reductions in antiviral activity against non-GT1 NS5B chimeric replicons compared to that against the GT1b subgenomic replicon. Smaller reductions in susceptibility, ranging from 0.2- to 33-fold, were observed for the inhibitor binding to the NNI1 (benzimidazole) site. The inhibitor binding to the NNI4 (benzofuran) site showed broad-spectrum antiviral activity against all chimeric replicons evaluated in this study. In conclusion, evaluation of HCV NNIs against intergenotypic chimeric replicons showed differences in activity spectrum for inhibitors that target different regions of the enzyme, some of which could be associated with specific residues that differ between GT1 and non-GT1 polymerases. Our study demonstrates the utility of chimeric replicons for broad-spectrum activity determination of HCV inhibitors.

In vitro resistance study of AG-021541, a novel nonnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA polymerase.

A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 A away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 muM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.

Novel chimeric genotype 1b/2a hepatitis C virus suitable for high-throughput screening.

A major obstacle in hepatitis C virus (HCV) research has been the lack of a permissive cell culture system that produces infectious viral particles. Significant breakthroughs have been achieved lately in establishing such culture systems. Yet to date, there are no reports of the applications of any of these systems in HCV drug screening. Here, we report the generation of two monocistronic, chimeric genotype 1 full-length HCV genome molecules. These molecules, C33J-Y835C-UBI and C33J-Y835C-FMDV2A, both contain the structural protein region from genotype 1 (subtype 1b, Con1) and the remaining region from the genotype 2a (JFH1) clone. Both contain the humanized Renilla luciferase reporter gene which is separated from the rest of the HCV open reading frame by two different cleavage sites. The viral RNAs replicated efficiently in transfected cells. Viral particles produced were infectious in naïve Huh7.5 cells, and the infectivity could be blocked by monoclonal antibody against a putative HCV entry cofactor, CD81. A pilot high-throughput screen of 900 unknown compounds was executed by both the genotype 2a subgenomic replicon system and the infectious system. Thirty-one compounds were identified as hits by both systems, whereas 78 compounds were identified as hits only for the infectious system, suggesting that the infectious system is capable of identifying inhibitors targeting the viral structural proteins and steps involving them in the viral life cycle. The infectious HCV system developed here provides a useful and versatile tool which should greatly facilitate the identification of HCV inhibitors currently not identified by the subgenomic replicon system.

Development of a novel dicistronic reporter-selectable hepatitis C virus replicon suitable for high-throughput inhibitor screening.

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neo(r) marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated Z' value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 microM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals > or =50 and < or =40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.

Identification and structure-based optimization of novel dihydropyrones as potent HCV RNA polymerase inhibitors.

A novel class of non-nucleoside HCV NS5B polymerase inhibitors has been identified from screening. A co-crystal structure revealed an allosteric binding site in the protein that required a unique conformational change to accommodate inhibitor binding. Herein we report the structure-activity relationships (SARs) of this novel class of dihydropyrone-containing compounds that show potent inhibitory activities against the HCV RNA polymerase in biochemical assays.