Inhibition of miR-214 expression by small molecules alleviates head and neck cancer metastasis by targeting ALCAM/TFAP2 signaling.

Predominantly, head and neck cancer (HNC) is considered a regional disease and develops in the nasal cavity, oral cavity, tongue, pharynx, and larynx. In the advanced stage, the HNC spread into distant organs. By the time head and neck cancer diagnosed, the estimated metastasis is occurred in 10-40% cases. The most important vital organs affected by distant metastasis are the lungs, bones, and liver. Despite several advancements in chemotherapies, no significant changes are observed as 5-year survival rate remains the same. Therefore, it is crucial to decipher molecular mechanisms contributing to the metastatic dissemination of head and neck cancer. Here, we tested a novel ALCAM/TFAP2 signaling by targeting multidisciplinary miR-214 expression in head and cancer cells. Our results revealed that HNC cell lines (CAL27, SCC-9, SCC-4, and SCC-25) exhibit higher expression of miR-214 compared with normal human bronchial epithelial (NHBE) cells. Higher expression of miR-214 drives the invasive potential of these cell lines. Down-regulation of miR-214 in CAL27 and SCC-9 cells either using an anti-miR-214 inhibitor (50nM) or a small molecule of green tea (EGCG) inhibited cell invasion. Treating CAL27 and SCC-9 cells with EGCG also reduces ALCAM expression, a key activated leukocyte cell adhesion molecule, potentially blocking mesenchymal phenotype. Dietary administration of EGCG significantly inhibits distant metastasis of SCC-9 cells into the lungs, liver, and kidneys. Our results also demonstrate that the reduction of miR-214 expression influences in vitro cell movement and extravasation, as evident by reduced CD31 expression, a neovascularization marker. Together, these studies suggest that identifying bioactive molecules that can inhibit distant metastasis regulated by the miRNAs may provide potent interventional approaches and a better understanding of the complex functions of miRNAs and their therapeutic targets for clinical application.

Differentially deregulated microRNAs contribute to ultraviolet radiation-induced photocarcinogenesis through immunomodulation: An-analysis of microRNAs expression profiling.

MicroRNAs (miRNAs) are short non-coding RNA molecules (18-25 nucleotides) that regulate several fundamental biological processes. Emerging evidence has shown more than 1500 miRNAs functions in the cell cycle, proliferation, apoptosis, oxidative stress, immune response, DNA damage, and epigenetics alterations. miRNAs are bidirectionally in nature and act as a tumor suppressor and as an oncogene through crosstalk between tumor cells and immune cells. Although the roles of miRNAs in several cancers are well studied, little is known about ultraviolet B (UVB) radiation-induced skin cancer. Here, we performed a comprehensive screening of 1281 miRNAs in tumor tissues and compared their expression with normal skin. Our results demonstrate that the expression levels of 587 miRNAs were altered in tumor tissues compared to their expression in normal skin. The expression of 337 miRNAs was upregulated from 1.5-12 folds, while the expression of 250 miRNAs was downregulated up to 1.5-10 folds in tumors. Further, intraperitoneal injection of a mimic of down-regulated miR-15b (30nM) and an inhibitor of upregulated miR-133a (20nM) protect UVB-induced suppression of contact hypersensitivity (CHS) response. In conclusion, we identified a network of altered miRNAs in tumors that can serve as prognostic biomarkers and therapeutic targets to manage photocarcinogenesis effectively.

Epigallocatechin Gallate (EGCG), an Active Phenolic Compound of Green Tea, Inhibits Tumor Growth of Head and Neck Cancer Cells by Targeting DNA Hypermethylation.

Head and neck cancers are among the deadliest cancers, ranked sixth globally in rates of high mortality and poor patient prognoses. The prevalence of head and neck squamous cell carcinoma (HNSCC) is associated with smoking and excessive alcohol consumption. Despite several advances in diagnostic and interventional methods, the morbidity of subjects with HNSCC has remained unchanged over the last 30 years. Epigenetic alterations, such as DNA hypermethylation, are commonly associated with several cancers, including HNSCC. Thus, epigenetic changes are considered promising therapeutic targets for chemoprevention. Here, we investigated the effect of EGCG on DNA hypermethylation and the growth of HNSCC. First, we assessed the expression levels of global DNA methylation in HNSCC cells (FaDu and SCC-1) and observed enhanced methylation levels compared with normal human bronchial epithelial cells (NHBE). Treatment of EGCG to HNSCC cells significantly inhibited global DNA hypermethylation by up to 70-80% after 6 days. Inhibition of DNA hypermethylation in HNSCC cells was confirmed by the conversion of 5-methylcytosine (5-mc) into 5-hydroxy methylcytosine (5hmC). DNA methyltransferases regulate DNA methylation. Next, we checked the effect of EGCG on the expression levels of DNA methyltransferases (DNMTs) and DNMT activity. Treatment of EGCG to HNSCC cells significantly reduced DNMT activity to 60% in SCC-1 and 80% in FaDu cells. The protein levels of DNMT3a and DNMT3b were downregulated in both cell lines after EGCG treatment. EGCG treatment to HNSCC cells reactivated tumor suppressors and caused decreased cell proliferation. Our in vivo study demonstrated that administration of EGCG (0.5%, w/w) as a supplement within an AIN76A diet resulted in inhibition of tumor growth in FaDu xenografts in nude mice (80%; p < 0.01) compared with non-EGCG-treated controls. The growth inhibitory effect of dietary EGCG on the HNSCC xenograft tumors was associated with the inhibition of DNMTs and reactivation of silenced tumor suppressors. Together, our study provides evidence that EGCG acts as a DNA demethylating agent and can reactivate epigenetically silenced tumor suppressors to inhibit the growth of HNSCC cells.

Japanese Encephalitis Virus NS4A Protein Interacts with PTEN-Induced Kinase 1 (PINK1) and Promotes Mitophagy in Infected Cells.

The nonstructural protein 4A (NS4A) of flaviviruses has been implicated as a "central organizer" of the membrane-bound replication complex during virus replication. However, its role in the host responses to virus infection is not understood. Using the yeast-two-hybrid library screen, we identified a multitude of host proteins interacting with the Japanese encephalitis virus (JEV) NS4A protein. Several of these interacting proteins are known to localize to the mitochondria. One of these proteins was PTEN-induced kinase 1 (PINK1), a serine/threonine-protein kinase known for its role in mitophagy. Here, we demonstrate the JEV-NS4A localization to the mitochondria and its interaction with PINK1 in Huh7 cells during JEV infection. The JEV-infected cells showed an enhanced mitophagy flux with a concomitant decline in the mitochondrial mass. We present data showing that JEV-NS4A alone was sufficient to induce mitophagy. Interference with mitochondrial fragmentation and mitophagy resulted in reduced virus propagation. Overall, our study provides the first evidence of mitochondrial quality control dysregulation during JEV infection, largely mediated by its NS4A protein. IMPORTANCE The JEV-infected mammalian cells show an enhanced mitophagy flux with a concomitant decline in the mitochondrial mass. We show that the NS4A protein of JEV localized to the mitochondria and interacted with PINK1 in Huh7 cells during infection with the virus and demonstrate that JEV-NS4A alone is sufficient to induce mitophagy. The study provides the first evidence of mitochondrial quality control dysregulation during JEV infection, largely mediated by its NS4A protein.

Regular Intake of Green Tea Polyphenols Suppresses the Development of Nonmelanoma Skin Cancer through miR-29-Mediated Epigenetic Modifications.

Previously, we and others have shown that the regular intake of green tea polyphenols (GTPs) reduces ultraviolet B (UVB) radiation-induced skin cancer by targeting multiple signaling pathways, including DNA damage, DNA repair, immunosuppression, and inflammation. Here, we determine the effect of GTPs on UVB-induced epigenetic changes, emphasizing DNA hypermethylation in UV-exposed skin and tumors and their association with miR-29, a key regulator of DNA methyltransferases (DNMTs). Skin cancer was induced in SKH-1 hairless mice following repeated exposures of UVB radiation (180 mJ/cm2, three times/week, 24 weeks) with or without GTPs supplementation (0.2%) in drinking water. Regular intake of GTPs inhibited tumor growth by hindering the cascade of DNA hypermethylation events. GTPs supplementation significantly blocked UVB-induced DNA hypermethylation in the skin (up to 35%; p < 0.0001) and in tumors (up to 50%; p < 0.0001). Experimental results showed that the levels of DNA hypermethylation were higher in GTPs-treated mice than in the control group. The expressions of miR-29a, miR-29b, and miR-29c were markedly decreased in UV-induced skin tumors, and GTPs administration blocked UVB-induced miR-29s depletion. Furthermore, these observations were verified using the in vitro approach in human skin cancer cells (A431) followed by treatment with GTPs or mimics of miR-29c. Increased levels of miR-29 were observed in GTPs-treated A431 cells, resulting in increased TET activity and decreased DNA hypermethylation. In conclusion, UVB-mediated miR-29 depletion promotes DNA hypermethylation and leads to enhanced tumor growth by silencing tumor suppressors. Regular intake of GTPs rescued UVB-induced miR-29 depletion and prevented tumor growth by maintaining reduced DNA hypermethylation and activating tumor suppressors. Our observations suggest that miR-based strategies and regular consumption of GTPs could minimize the risk of UVB-induced skin cancers and contribute to better management of NMSCs.

Splicing regulator SRSF1-3 that controls somatic hypermutation of IgV genes interacts with topoisomerase 1 and AID.

Somatic hypermutation (SHM) of Ig genes is initiated by activation-induced cytidine deaminase (AID) and requires target gene transcription. A splice isoform of SRSF1, SRSF1-3, is necessary for AID-dependent SHM of IgV genes. Nevertheless, its exact molecular mechanism of action in SHM remains unknown. Our in silico studies show that, unlike SRSF1, SRSF1-3 lacks a strong nuclear localization domain. We show that the absence of RS domain in SRSF1-3 affects its nuclear localization, as compared to SRSF1. Consequently, SRSF1-3 is predominantly present in the cytoplasm. Remarkably, co-immunoprecipitation studies showed that SRSF1-3 interacts with Topoisomerase 1 (TOP1), a crucial regulator of SHM that assists in generating ssDNA for AID activity. Moreover, the immunofluorescence studies confirmed that SRSF1-3 and TOP1 are co-localized in the nucleus. Furthermore, Proximity Ligation Assay corroborated the direct interaction between SRSF1-3 and TOP1. An interaction between SRSF1-3 and TOP1 suggests that SRSF1-3 likely influences the TOP1 activity and consequently can aid in SHM. Accordingly, SRSF1-3 probably acts as a link between TOP1 and SHM, by spatially regulating TOP1 activity at the Ig locus. We also confirmed the interaction between SRSF1-3 and AID in chicken B-cells. Thus, SRSF1-3 shows dual-regulation of SHM, via interacting with AID as well as TOP1.

Regulation of Monoamine Oxidase B Gene Expression: Key Roles for Transcription Factors Sp1, Egr1 and CREB, and microRNAs miR-300 and miR-1224.

Monoamine oxidase B (MAO-B), a flavoenzyme located in the outer mitochondrial membrane, is involved in the catabolism of monoamines. Altered levels of MAO-B are associated with cardiovascular/neuronal diseases. However, molecular mechanisms of MAO-B gene regulation are partially understood. We undertook a systematic analysis of the MAO-B gene to identify the key transcriptional/post-transcriptional regulatory molecules. Expression of MAO-B promoter-reporter constructs in cultured cells identified the -144/+25-bp domain as the core promoter region. Stringent in silico analysis of this core promoter predicted binding sites for several transcription factors. Over-expression/down-regulation of transcription factors Sp1/Egr1/CREB increased/decreased the MAO-B promoter-reporter activity and endogenous MAO-B protein level. Electrophoretic mobility shift assays and ChIP assays provided evidence for interactions of Sp1/Egr1/CREB with the MAO-B promoter. MAOB transcript level also positively correlated with the transcript level of Sp1/Egr1/CREB in various human tissue samples. Computational predictions using multiple algorithms coupled with systematic functional analysis revealed direct interactions of the microRNAs miR-1224 and miR-300 with MAO-B 3'-UTR. Dopamine dose-dependently enhanced MAO-B transcript and protein levels via increased binding of CREB to MAO-B promoter and reduced miR-1224/miR-300 levels. 8-Bromo-cAMP and forskolin augmented MAO-B expression, whereas inhibition of PKA diminished the gene expression suggesting involvement of cAMP-PKA axis. Interestingly, Sp1/Egr1/CREB/miR-1224 levels correlate with MAO-B expression in rodent models of hypertension/MPTP-induced neurodegeneration, indicating their roles in governing MAO-B gene expression in these disease states. Taken together, this study elucidates the previously unknown roles of the transcription factors Sp1/Egr1/CREB and microRNAs miR-1224/miR-300 in regulating MAO-B gene expression under basal/disease states involving dysregulated catecholamine levels.

Cytotoxic and Genotoxic effect on the Buccal Mucosa Cells of Patients Undergoing Fixed Orthodontic Treatment.

AIM: To evaluate the presence of metal ions and deoxyribonucleic acid damage on the cells of buccal mucosa in subjects scheduled to undergo fixed orthodontic treatment. MATERIALS AND METHODS: Eighty patients scheduled to undergo orthodontic treatment were included in the present study. Samples were collected from buccal mucosa of the subjects at five different intervals: before the starting of the fixed appliance therapy, 5 months after the insertion of the appliance, 10 months after insertion of the appliance, 15 months after insertion of the appliance and 20 months after insertion of the appliance. Flow cytometry was further used for assessment of apoptosis. Comet assay was used for evaluating the metal ions associated deoxyribonucleic acid ((DNA) damage of buccal epithelial cells. Atomic absorption spectrometry was used for measuring the nickel (Ni), chromium (Cr) and zinc (Zn) levels in the cells of the buccal mucosa. Analysis of data was done by SPSS software version 16.0. RESULTS: A significant increase in the Ni, Cr and Zn concentra -tion during orthodontic treatment was observed. A progressive non-significant decrease in the percentage of viable cells from a baseline value to the end of the treatment was observed. A significant increase in the head diameter, DNA in tail and tail length, starting from the pretreatment value to the end of orthodontic treatment, was also observed. CONCLUSION: Timely checking of deoxyribonucleic acid (DNA) damage and nuclear changes should be done for detecting earlier adverse changes. CLINICAL SIGNIFICANCE: In patients wearing orthodontic appliances, no clinical impact occurs by wearing fixed appliances.

Pendulum Therapy of Molar Distalization in Mixed Dentition.

Early and timely pedo-orthodontic treatment is aimed at eliminating the disturbances of skeletal or dentoalveolar development, to harmonize the stomatognathic system before the full eruption of all permanent teeth. The advantages of pendulum appliance are its minimal dependence on patient's compliance (child cooperation), ease of fabrication, onetime activation and adjustment of the springs if necessary to correct minor transverse and vertical molar positions. This article reports a successful treatment method of class II malocclusion with pendulum appliance in mixed dentition phase. Distalization of maxillary molar was done, followed by guidance of canine impaction orthodontically and other dental correction using 0.022 MBT appliances. Posttreatment results were stable and remarkable. How to cite this article: Patil RU, Prakash A, Agarwal A. Pendulum Therapy of Molar Distalization in Mixed Dentition. Int J Clin Pediatr Dent 2016;9(1):67-73.

Reinforced tension line suture closure after midline laparotomy in emergency surgery.

Midline laparotomy is an emergency surgical operation frequently performed in cases of intra-abdominal pathology. Closure of the incision is usually done by continuous suturing by mass closure. In an emergency operation the intra-abdominal milieu is usually contaminated leading to gut oedema and, hence, an increase in postoperative intra-abdominal pressure. It is complicated by wound dehiscence, burst abdomen, etc. The cause of this complication is an increase in horizontal tensile forces on the site of the insertion of sutures which cuts the sheath. In this technique of reinforced tension line suture peak tensile forces are distributed from the suture base to the surrounding tissue through a horizontal suture, thereby preventing the suture from cutting through the tissue. From July 2007 to June 2009 patients requiring laparotomy were randomly divided into test and control groups by a 'closed envelope' technique. Their postoperative intra-abdominal pressure was recorded by urinary bladder catheter manometry. The result of this technique was compared with the incidence of burst abdomen in cases where it was closed by continuous suture. A total of 190 patients underwent laparotomy. In 90 the abdomen was closed by reinforced tension line (RTL) and in 100 patients by continuous suturing. None of the RTL group had a burst abdomen. Thirteen who had closure by continuous suture had a burst abdomen. The analysis of the results was done using the chi-square test. On comparing the incidence of burst abdomen in cases operated by continuous suture technique and by RTL, the P value was found to be 0.0026 which is highly significant. On analysis of the incidence of burst abdomen in cases having a grade II intra-abdominal pressure the P value was found to be 0.0009 which is highly significant. Closure of midline incision by RTL reduces the incidence of burst abdomen. Registration No. PROVCTRI/2008/091/000269 (http://www.ctri.in).

Congenital intra-mesosigmoid hernia- a case report of a rare sigmoid mesocolon hernia.

Internal hernia is the protrusion of the viscera through normal or abnormal peritoneal or mesenteric apertures within the confines of peritoneal cavities.

Doctor patient communication-a vital yet neglected entity in Indian medical education system.

Doctor patient communication is the most important and an integral part of any treatment regimen. Properly carried out it has been shown to have a therapeutic effect equivalent to drugs. Despite being so important part of treatment it is more than often taken and carried out casually. Apart from apathy towards this practice, its omission in the medical study curriculum is an important factor. This study was carried in amongst the surgical residents of surgical departments of various medical colleges to assess the attitude of surgical residents towards patient doctor communication. A questionnaire was forwarded by mail and email and the response was assessed: The responses of the surgical residents from various residents from different medical colleges were similar. Most of the residents prefer inclusion of communication skill in medical education curriculum.

Sacrococcygeal masses other than meningomyelocele.

This series comprises of a variety of sacrococcygeal masses other than meningomyelocele that presented to the department of pediatric surgery of Medical College Kolkata over last 10 years. In this series, 23 cases of sacrococcygeal masses are included. Barring meningomyelocele, teratoma constitutes a major group of cases. It also includes few other interesting and atypical masses such as presacral dermoid, degenerated nerve fiber, fibrofatty tissue, rhabdomyosarcoma, etc. This is an endeavor to enlighten ourselves so that the diagnosis and management of unfamiliar sacrococcygeal masses can be done.

Large neurofibroma of trunk.

UNLABELLED: Neurofibroma is a benign tumor of cutaneous nerves. These are benign tumors which may have a varied presentations ranging from a cosmetic problem to a spinal tumor which may lead to neural complications. We here by present a case where the patient has become an appendage of the tumor and its the mass of the tumor that has handicapped the patient. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12262-010-0129-x) contains supplementary material, which is available to authorized users.

Double gall bladder-a rare entity.

Cholecystectomy is the most commonly performed operation in surgery. Variations inanatomical disposition are not infrequent. However variations in number of cystic ductand gall bladder is quiet rare. This poses a diagnostic and management problem withcomplications during surgery and missed gall bladder being reported in world literature. We here by report a case of double gall bladder with double cystic duct that was managed by laparoscopic surgery.

S-phase fraction and DNA ploidy in oral leukoplakia.

BACKGROUND: The risk of malignant conversion in oral leukoplakia is well documented. Histological findings are often unreliable and it is difficult to predict on the basis of clinical and histopathological changes which leukoplakic lesion will turn malignant. METHODS: We used the technique of flow cytometry to evaluate the ploidy status, DNA index and S-phase fraction in leukoplakia, oral cancer and normal oral mucosal biopsies and compared it with histological findings. The study was carried out on 30 patients with oral cancer, 60 with leukoplakia and 30 with normal oral mucosal biopsies. RESULTS: The aneuploidy rate in oral cancers was 64%, for leukoplakia 20%, while all normal mucosal biopsies were diploid. Aneuploid lesions also had a greater S-phase fraction (SPF). The DNA Index (DI) of aneuploid oral cancers was 1.72 and aneuploid leukoplakias was 1.24. Leukoplakia specimens which showed histological evidence of dysplasia had aneuploidy rate of 38%, DI of 1.19 and SPF of 6.2%. The corresponding values for leukoplakia specimens without dysplasia were 14%, 1.09 and 4.1%, respectively. CONCLUSION: The method of flow cytometry can be used to identify the subset of leukoplakia patients who are at a higher risk of malignant conversion. These patients could undergo more rigid surveillance or undergo excision biopsy of their lesions.

Radiographic griding of subcutaneous soft tissue metallic foreign bodies in emergency department.

Radiographic grid for localization of soft tissue metallic foreign bodies is a modification of traditional radiography. Twenty localization procedures using simple radiographic grid was successfully performed. Its low cost and easy to perform makes it a useful tool in emergency setting.

Pleuropulmonary blastema with cystic nephroma - A rare presentation and surgical dilemma.

Pleuropulmonary balstema with cystic nephroma is a rare dual pathology of pediatric age group. The etiopathogenesis of this entity is not known, still researches indicate towards a common genetic cause. We report a case of this dual pathology in a one and half year old male. Till now only 5 cases have been reported.

Improvised laparoscopic stone picker.

Laparoscopic cholecystectomy is the treatment of choice for calculus cholecystitis. It is frequently complicated by stone spillage and retrieval of these stones is a cumbersome process which is frequently complicated by injuries. Moreover left over stones frequently leads to complication. We have hereby improvised a laparoscopic hand instrument which quickly and easily recovers intraabdominal free and scattered gall stones with a loss of little bit of intra-abdominal CO(2). This stone picker was applied in over 50 cases with successful recovery of stones and a mean loss of about 0.1 Lts of carbon dioxide gas.