Mucin-4: A novel marker for oral cancer.

BACKGROUND: Mucins are high-molecular-weight glycoproteins and are implicated in diverse biological functions. MUC4, a member of transmembrane mucin family, is expressed in airway epithelial cells and body fluids such as the saliva, tear film, ear fluid and breast milk. In addition to its normal expression, an aberrant expression of MUC4 has been reported in a variety of carcinomas. Till date, very few studies have discussed about MUC4 expression in normal and cancerous oral mucosa. AIM: The aim of the study is to evaluate the expression of MUC4 in varying grades of oral squamous cell carcinoma (OSCC) and also to analyze its role played in oral carcinogenesis. MATERIALS AND METHODS: Formalin-fixed paraffin-embedded tissues of five cases of normal tissue, ten cases of well-differentiated OSCC, ten cases of moderately differentiated OSCC and ten cases of poorly differentiated OSCC were retrieved from the archives of the department, and MUC4 antigen was immunohistochemically localized. STATISTICAL ANALYSIS: The result was subjected to statistical analysis using Pearson's Chi-square test and one-way analysis of variance. RESULTS: About 70% of OSCC were stained positive with MUC4 antigen. Maximum intensity of staining was noted in well-differentiated OSCC. A steady decrease in MUC4 staining was noted with the increase in histological grading of OSCC. CONCLUSION: The findings of the study suggest that MUC4 plays a vital role in the pathogenesis of OSCC and can be regarded as a novel marker for OSCC.

Effect of pH on solubility of white Mineral Trioxide Aggregate and Biodentine: An in vitro study.

Background. The aim of this study was to evaluate the effect of acidic, neutral and alkaline environments on the solubility of white mineral trioxide aggregate (WMTA) and Biodentine (BD). Methods. Thirty-nine ring molds were randomly divided into three groups of A, B, and C (n = 12) with pH values of 7.4, 4.4 and 10.4, respectively, and an empty mold was used as a control. Each group was further divided into two subgroups (1 and 2) according to the material studied. The samples in groups A, B and C were transferred into synthetic tissue fluid buffered at pH values of 7.4, 4.4 and 10.4, respectively, and kept in an incubator at 37°C with 100% humidity. Daily solubility at 1-, 2-, 5-, 14-, 21-, and 30-day intervals and cumulative solubility up to 5-, 14-, and 30-day intervals were calculated. Statistical analysis was carried out with independent-samples t-test, two-way ANOVA and post hoc Tukey tests using SPSS 18. Statistical significance was set at P<0.05. Results. Both WMTA and BD exhibited the highest solubility in acidic pH with 5.4235±0.1834 and 10.7516±0.0639 mean cumulative solubility values at 30-day interval, respectively. At all exposure times, BD was significantly more soluble than WMTA (P<0.001). Conclusion. Acidic periapical environment jeopardized the solubility of both WMTA and BD, affecting their sealing characteristics in clinical applications like perforation repair procedures and blunderbuss canals.

Methyl group reorientation under ligand binding probed by pseudocontact shifts.

Liquid-state NMR spectroscopy is a powerful technique to elucidate binding properties of ligands on proteins. Ligands binding in hydrophobic pockets are often in close proximity to methyl groups and binding can lead to subtle displacements of methyl containing side chains to accommodate the ligand. To establish whether pseudocontact shifts can be used to characterize ligand binding and the effects on methyl groups, the N-terminal domain of HSP90 was tagged with caged lanthanoid NMR probe 5 at three positions and titrated with a ligand. Binding was monitored using the resonances of leucine and valine methyl groups. The pseudocontact shifts (PCS) caused by ytterbium result in enhanced dispersion of the methyl spectrum, allowing more resonances to be observed. The effects of tag attachment on the spectrum and ligand binding are small. Significant changes in PCS were observed upon ligand binding, indicating displacements of several methyl groups. By determining the cross-section of PCS iso-surfaces generated by two or three paramagnetic centers, the new position of a methyl group can be estimated, showing displacements in the range of 1-3 Å for methyl groups in the binding site. The information about such subtle but significant changes may be used to improve docking studies and can find application in fragment-based drug discovery.

Genetic variations of IL-10: Identification of novel variations and evaluation of the impact of the SNPs/haplotype in the promoter region with the progression of Oral Squamous Cell Carcinoma in Indian population.

The correlation of interleukin 10 (IL-10) with the outbreak and progression of cancer has been well established as it contributes to tumor immune evasion. Convincing number of evidences has been accumulated to reflect the critical correlation between IL-10 polymorphism and tumorogenesis. Several polymorphic sites at promoter regions have been reported to be associated with cancer susceptibility. The purpose of this study was to examine the effect of modulated genotypes in the promoter region of IL-10 gene with life-style habits in oral squamous cell carcinoma (OSCC) in the Indian population. A total of 300 subjects (100 OSCC, 50 precancer and 150 healthy controls) were recruited in this study. The IL-10 promoter region was amplified in 14 overlapping fragments by PCR and further screened through the high throughput technique of denaturing high-performance liquid chromatography (dHPLC) followed by sequencing. We identified three novel variations at positions (-924, -1045 & -1066); we also found some known SNPs (-592C/A, -657G/A, -851G/A, -819C/T, -1082A/G). The identified novel variations were submitted to the NCBI Gene Bank (accession numbers KT153594, KT291742 and KT291743). We also noticed a significant association of polymorphisms (-592C/A, -819C/T and -1082A/G) individually as well as in combination (haplotypes) along with lifestyle habits for the risk of oral carcinoma (p<0.0001). We have reported three novel SNPs in the Indian population for the first time, and these SNPs may be associated with OSCC. Besides, we showed the first evidence of IL-10 haplotypes, i.e., CCG and CTG, may act as a biomarker for early detection of oral pre-cancerous/cancerous lesions or treatment management of oral carcinoma.

Micronucleus Assay: An Early Diagnostic Tool to Assess Genotoxic Changes in Patients with Tobacco Use, Oral Leukoplakia and Oral Submucous Fibrosis.

INTRODUCTION: Micronuclei (MNi) are acentric chromatid or chromosome fragments produced via genetic damage through genotoxic agents contained in tobacco and betel nut. Evidently, the various Oral Potentially Malignant Disorders (OPMDs) like oral lichen Planus, oral leukoplakia and Oral Submucous Fibrosis (OSMF) demonstrate MNi, as a substantiation of genetic damage. As these changes can be easily appreciated in oral exfoliated cells, an exfoliated cell based MNi assay might be utilized as handy and non invasive biomonitoring tool for gauging the genetic damage and hence the propensity for malignant transformation in OPMDs. To this end, MNi are definitely easier to evaluate when compared to chromosome aberrations. AIM: To compare the MNi frequency in normal mucosa, in individuals using various tobacco forms without oral leukoplakia, individuals using various tobacco forms with oral leukoplakia, and areca nut chewers with OSMF, using three different stains. MATERIALS AND METHODS: Oral exfoliated cells from 50 cases of normal mucosa (Group I), 50 cases of tobacco chewing people without Oral Leukoplakia (Group II), 50 cases of people with Oral Leukoplakia (Group III) and 50 cases of areca nut chewers with OSMF (Group IV) were taken. MNi frequencies were compared in these groups using three different stains i.e., Papanicolaou (PAP) stain, May Grunwald Giemsa (MGG) stain and Feulgen stain. The data between cases (Group II, III and IV) and control groups (Group I) was analyzed by Kruskal-Wallis Test. The comparison between two independent groups was done by Mann-Whitney U test and interstain comparison between cases and control was done by Wilcoxon Signed Rank Test and the individual p-value was obtained. RESULTS: A significant increase in the count was observed during transition of normal mucosa to OPMDs. The best stain for detecting MNi was PAP stain followed by MGG stain and Feulgen stain. CONCLUSION: The higher mean MNi count for PAP stain and MGG stain could be attributed to nonspecific staining. Further study using a larger sample size on quantitative assessment of MNi count in various OPMDs is warranted.

Impacts of TNF-LTA SNPs/Haplotypes and Lifestyle Factors on Oral Carcinoma in an Indian Population.

BACKGROUND AND OBJECTIVES: To investigate a potential association between single-nucleotide polymorphisms (SNPs) and  haplotypes at the TNFA-LTA locus and the development of oral cancer in an Indian population. MATERIALS AND METHODS: In this study, 150 oral precancer/cancer samples (50 precancer and 100 cancer), along with an equal number of control samples, were genotyped. Six SNPs at the TNF-LTA locus (i.e., -238G/A, -308G/A, -857C/T, -863C/A, -1031T/C, and +252A/G) were analyzed by use of a polymerase chain reaction-restriction fragment length polymorphism method, the assay was validated by sequencing 10 % of samples. RESULTS: The allelic frequencies of TNFA and LTA SNPs were found to be significantly associated with the risk of oral cancer and precancerous lesions in comparison with controls (P < 0.0003). Further haplotypic analysis showed that two haplotypes (ATCTGG and ACACGG) served as risk haplotypes for oral cancer. These haplotypes were also found to be significantly and positively associated with lifestyle habits (tobacco chewing P = 0.04, odds ratio [OR] 3.4) and socioeconomic status (P = 0.01, OR 3.4). We noticed an increased percentage of risk haplotypes correlating with the aggressiveness of oral cancer. The percentages of risk haplotypes were found to be threefold higher in precancer and fourfold higher in advanced stages of oral cancer in comparison with controls. CONCLUSION: Five SNPs at the TNF-LTA locus (i.e., -308G>A, -857C>T, -863C>A, -1031T>C, and +252A>G) were found to be associated with the development of oral cancer. Two haplotypes (ATCTGG and ACACGG) emerged as major risk haplotypes for oral carcinoma progression and were also found to be associated with lifestyle factors and clinical aggressiveness. These findings make the TNF-LTA locus a suitable candidate for a future biomarker, which may be used either for early detection or for helping to improve treatment efficacy and effectiveness.

Proline Conformation in a Functional Tau Fragment.

The conformational state of distinct prolines can determine the folding of a protein but equally other biological processes when coupled to a conformation-sensitive secondary reaction. For the neuronal tau protein, the importance of proline conformation is underscored by its interaction with different prolyl cis/trans isomerases. The proline conformation would gain even further importance after phosphorylation of the preceding residue by various proline-directed kinases. A number of molecular diseases including Alzheimer's disease and traumatic brain injury were thereby recently qualified as "cistauosis", as they would imply a cis conformation for the pThr231-Pro232 prolyl bond. We here investigate by NMR spectroscopy the conformation of all prolines in a functional Tau fragment, Tau[208-324]. Although we can detect and identify some minor conformers in the cis form, we show that all prolines are for over 90% in the trans conformation. Phosphorylation by CDK2/CycA3, which notably leads to complete modification of the Thr231 residue, does not change this conclusion. Our data hence disagree with the notion that specific prolyl bonds in tau would adopt preferentially the cis conformation.

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro(314)-Trp(316) turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro(314) of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function.

H/D exchange of a 15N labelled Tau fragment as measured by a simple Relax-EXSY experiment.

We present an equilibrium H/D exchange experiment to measure the exchange rates of labile amide protons in intrinsically unfolded proteins. By measuring the contribution of the H/D exchange to the apparent T1 relaxation rates in solvents of different D2O content, we can easily derive the rates of exchange for rapidly exchanging amide protons. The method does not require double isotope labelling, is sensitive, and requires limited fitting of the data. We demonstrate it on a functional fragment of Tau, and provide evidence for the hydrogen bond formation of the phosphate moiety of Ser214 with its own amide proton in the same fragment phosphorylated by the PKA kinase.

Effectiveness of four different final irrigation activation techniques on smear layer removal in curved root canals : a scanning electron microscopy study.

OBJECTIVE: The aim of this study was to assess the efficacy of apical negative pressure (ANP), manual dynamic agitation (MDA), passive ultrasonic irrigation (PUI) and needle irrigation (NI) as final irrigation activation techniques for smear layer removal in curved root canals. MATERIALS AND METHODS: Mesiobuccal root canals of 80 freshly extracted maxillary first molars with curvatures ranging between 25° and 35° were used. A glide path with #08-15 K files was established before cleaning and shaping with Mtwo rotary instruments (VDW, Munich, Germany) up to size 35/0.04 taper. During instrumentation, 1 ml of 2.5% NaOCl was used at each change of file. Samples were divided into 4 equal groups (n=20) according to the final irrigation activation technique: group 1, apical negative pressure (ANP) (EndoVac); group 2, manual dynamic agitation (MDA); group 3, passive ultrasonic irrigation (PUI); and group 4, needle irrigation (NI). Root canals were split longitudinally and subjected to scanning electron microscopy. The presence of smear layer at coronal, middle and apical levels was evaluated by superimposing 300-µm square grid over the obtained photomicrographs using a four-score scale with X1,000 magnification. RESULTS: Amongst all the groups tested, ANP showed the overall best smear layer removal efficacy (p < 0.05). Removal of smear layer was least effective with the NI technique. CONCLUSION: ANP (EndoVac system) can be used as the final irrigation activation technique for effective smear layer removal in curved root canals.

Primary intraosseous mucoepidermoid carcinoma of maxilla.

Primary intraosseous mucoepidermoid carcinoma (PIOC) of the jaw bones is an extremely rare malignant salivary gland tumor, comprising 2-3% of all mucoepidermoid carcinomas reported. It is commonly seen in the posterior part of the mandible; its occurrence in the maxilla is rare. They have been reported in patients of all ages, ranging from 1 to 78 years, with the overwhelming majority occurring in the 4th and 5th decades of life. They are histologically low-grade cancers and radiographically seen as uniocular or multiocular lesions. We report a rare case of PIOC in posterior palatal region in 18-year-old male.

A new strategy for sequential assignment of intrinsically unstructured proteins based on 15N single isotope labelling.

We describe a new efficient strategy for the sequential assignment of amide resonances of a conventional (15)N-(1)H HSQC spectrum of intrinsically unfolded proteins, based on composite NOESY-TOCSY and TOCSY-NOESY mixing times. These composite mixing times lead to a Hα-proton mediated unidirectional transfer of amide to amide proton. We have implemented the composite mixing times in an HSQC-NOESY-HSQC manner to obtain directional connectivity between amides of neighbouring residues. We experimentally determine the optimal mixing times for both transfer schemes, and demonstrate its use in the assignment for both a fragment of the neuronal tau protein and for α-synuclein.

Long-lived states to monitor protein unfolding by proton NMR.

The relaxation of long-lived states (LLS) corresponds to the slow return to statistical thermal equilibrium between symmetric and antisymmetric proton spin states. This process is remarkably sensitive to the presence of external spins and can be used to obtain information about partial unfolding of proteins. We detected the appearance of a destabilized conformer of ubiquitin when urea is added to the protein in its native state. This conformer shows increased mobility in the C-terminus, which significantly extends the lifetimes of proton LLS magnetisation in Ser-65. These changes could not be detected by conventional measurements of T(1) and T(2) relaxation times of protons, and would hardly be sensed by carbon-13 or nitrogen-15 relaxation measurements. Conformers with similar dynamic and structural features, as revealed by LLS relaxation times, could be observed, in the absence of urea, in two ubiquitin mutants, L67S and L69S.

Aggressive fibromatosis of anterior maxilla.

Aggressive fibromatosis is a comparitively rare tumor with unpredictable growth and varying local recurrence rates. It does not develop distant metastases but locally it shows an aggressive and infiltrative behavior. Clinically, aggressive fibromatosis manifests as a painless, firm, often rapidly enlarging mass, fixed to underlying bone or soft tissue. It is never encapsulated. Histologically, it is rich in collagen and fibroblastic cells that are devoid of hyperchromatic or atypical nuclei, but with more variable cellularity in different tumor sections.

Mineralized components and their interpretation in the histogenesis of peripheral ossifying fibroma.

BACKGROUND: Peripheral ossifying fibroma (POF) is a lesion of gingival tissue that predominantly affects women and is usually located in maxilla, anterior to molars. The definitive diagnosis is established by histopathological examination, which reveals the presence of cellular connective tissue with focal calcifications. OBJECTIVE: This study hypothesizes the histogenesis of POF by analyzing the diverse spectrum of mineralized components with a polarizing microscope. MATERIALS AND METHODS: A retrospective study was undertaken which involved a detailed review of clinical, radiographic and histopathological features of 22 cases of POF, retrieved from departmental archives. These cases were subsequently stained with a histochemical stain (van Gieson) and observed under a polarizing microscope. RESULTS: The study revealed that the most common age of occurrence was in second and third decades with a strong female predilection (73%), Interdental papilla of the maxillary anterior region was the most commonly afflicted site. About 90% cases showed no radiographic features.Histopathological examination showed that 73% cases consisted of a fibrocellular connective tissue stroma surrounding the mineralized masses. 50% mineralized masses comprised of woven bone, 18% showed combination of lamellar bone and cellular cementum, 18% showed only cementum (cellular and acellular), and remaining 13.6% exhibited a mixture of woven and lamellar bone under polarizing microscope. CONCLUSION: The study supports the theory that POF develops from cells of periodontal ligament (PDL)/periosteum as undifferentiated mesenchymal cells having an inherent proliferative potential to form bone or cementum, whose nature can be confirmed by polarizing microscope.

Relevance of tumor angiogenesis patterns as a diagnostic value and prognostic indicator in oral precancer and cancer.

Tumor angiogenesis occurs by recruitment of endothelial cell precursors or by sprouting of existing capillaries, which differ from the normal vasculature by having an altered morphology that can be exploited for diagnosis and as a prognostic indicator. Improved technologies have propelled diagnosis into a new era. These technologies have to be used with great precision. The diagnosis of a dysplastic premalignant lesion of the oral mucosa cannot be based solely on clinical findings. Therefore histologic evaluation of a representative biopsy specimen is necessary. Accurate judgment of the proper site for biopsy is essential for reaching a correct diagnosis. The aim of this report is to analyze the vascular patterns with the help of direct oral microscopy and the technique of stereo-optical microscopy in the oral cavity to select biopsy sites, and compare the outcome of a directed biopsy with that of biopsy specimens obtained from sites selected solely on the basis of clinical criteria. The study sample comprised 50 oral mucosal lesions. A statistically significant difference was noted between samples judged to be microscopically representative sites. We conclude that this method would aid in early and better diagnosis and treatment planning of oral premalignant and malignant lesions by assessing the various vascular patterns in the mucosa.

Lymphangioma of cheek region-an unusual presentation.

BACKGROUND AND OBJECTIVES: Lymphangiomas are benign hamartomatous tumors of the lymphatic vessels. A lymphatic malformation is a congenital defect that occurs during early embryonic development when the lymphatic vessels do not properly form. The vessels may become blocked and enlarged as lymphatic fluid collects in the vessels, forming a mass or a cyst. About 50% of all lesions are noted at birth and around 90% develop by 2 years of age. MATERIALS AND METHODS: We report the case of lymphangioma, with an unusual presentation in the cheek region resulting into vestibular obliteration and expanding to the left maxilla diagnosed on the basis of histopathology. The review of literature concerning the clinical and histological features as well as the proper management concerning this hamartomatous entity is included. RESULTS AND CONCLUSION: The various treatment modalities for lymphangioma are surgical excision, radiation therapy cryotherapy, electrocautery, sclerotherapy, steroid administration, embolization, ligation, laser surgery with Nd-YAG and CO2 and radiofrequency tissue ablation technique. An early diagnosis and intervention helps in reducing the functional, psychological disturbances and cosmetic disfigurement.