Predicting novel candidate human obesity genes and their site of action by systematic functional screening in Drosophila.

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action.

Application of Value Framework to Phase III Trials of Immune Checkpoint Inhibitors in Esophageal and Gastric Cancer.

BACKGROUND: Recent trials testing immune-checkpoint inhibitors in esophago-gastric malignancies have shown mixed results. We aim to assess key subgroups using the ASCO Net Health Benefit Score (NHBS) and ESMO Magnitude of Clinical Benefit Scale (MCBS). MATERIALS AND METHODS: A search for phase III trials of FDA-approved anti-PD-1 or anti-PD-L1 drugs in esophago-gastric cancer trials was identified using www.clinicaltrials.gov. These published studies were scored using the ASCO NHBS and ESMO MCBS. The ASCO NHBS scores were compared by primary site of cancer (esophageal vs gastric) and PD-L1 expression using the Mann-Whitney test and the ESMO-MCBS grading, by Fisher's Exact test. RESULTS: Fifteen of 45 clinical trials were included. Of them, 6 were primarily esophageal cancer trials, and 9 were primarily gastric cancer trials. Ten stratified their analysis based on PD-L1 expression. The ASCO NHBS score was higher (mean 40, range 20 to 56.6 vs. mean 12, range -1.1 to 18.4, P < .01) for esophageal cancer than gastric cancer. No difference was observed in survival and response endpoints between the 2 groups. Similarly, the ESMO MCBS scored higher for esophageal cancer group than gastric cancer (P < .05). Additionally, the scores were higher in those with high PD-L1 expression vs. low PD-L1 (mean 36, range 11.2-66.6 vs. mean 14, range -19.5 to 43.6, P < .05). CONCLUSION: The ASCO NHB and ESMO scores were consistently higher among esophageal cancer trials than gastric cancer trials and in those with high PD-L1 expression than low expression. Histology and PD-L1 expression should be considered when discussing value of immunotherapy to patients.

Functional interactions between posttranslationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans.

The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.

Profiling rhythmicity of bile salt hydrolase activity in the gut lumen with a rapid fluorescence assay.

Diurnal rhythmicity of cellular function is key to survival for most organisms on Earth. Many circadian functions are driven by the brain, but regulation of a separate set of peripheral rhythms remains poorly understood. The gut microbiome is a potential candidate for regulation of host peripheral rhythms, and this study sought to specifically examine the process of microbial bile salt biotransformation. To enable this work, an assay for bile salt hydrolase (BSH) that could work with small quantities of stool samples was necessary. Using a turn-on fluorescence probe, we developed a rapid and inexpensive assay to detect BSH enzyme activity with concentrations as low as 6-25 µM, which is considerably more robust than prior approaches. We successfully applied this rhodamine-based assay to detect BSH activity in a wide range of biological samples such as recombinant protein, whole cells, fecal samples, and gut lumen content from mice. We were able to detect significant BSH activity in small amounts of mouse fecal/gut content (20-50 mg) within 2 h, which illustrates its potential for use in various biological/clinical applications. Using this assay, we investigated the diurnal fluctuations of BSH activity in the large intestine of mice. By using time restricted feeding conditions, we provided direct evidence of 24 h rhythmicity in microbiome BSH activity levels and showed that this rhythmicity is influenced by feeding patterns. Our novel function-centric approach has potential to aid in the discovery of therapeutic, diet, or lifestyle interventions for correction of circadian perturbations linked to bile metabolism.

LovesCompany: evaluating the safety and feasibility of a mental health-focused online community for adolescents.

Background: Adolescents are at risk for mental health (MH) disorders but are unlikely to seek services and may be reluctant to talk about their MH. An anonymous, online MH-focused community could help reduce suffering. However, online forums can also promote negative behaviours such as cyberbullying. This study aimed to evaluate the safety and feasibility of an online community - LovesCompany - to improve MH outcomes for adolescents.Methods: American adolescents (14-17 years) were recruited through social media. Eligible participants were randomised to LovesCompany or a placebo MH resource site. Outcomes were assessed every other week for six months, and at twelve months. Multilevel models assessed group differences in depression, anxiety, and suicidal ideation. A subgroup of participants participated in qualitative interviews.Results: Participants (N = 202) were mostly female (70%), White non-Hispanic (69%), and cisgender (80%). There were no instances of inappropriate behaviour such as bullying or posting explicit content. Symptoms for both groups improved over time. Participants appreciated hearing others' experiences and valued the opportunity to offer support.Conclusion: Although adolescents are often resistant to MH treatment, they appear to be interested in anonymous, online, MH-focused conversation, and can benefit from giving and seeking support. Finding a balance between an appealing user experience, ethical considerations, and resource needs is challenging.

Anchoring alien chromosome segment substitutions bearing gene(s) for resistance to mustard aphid in Brassica juncea-B. fruticulosa introgression lines and their possible disruption through gamma irradiation.

KEY MESSAGE: Heavy doses of gamma irradiation can reduce linkage drag by disrupting large sized alien translocations and promoting exchanges between crop and wild genomes. Resistance to mustard aphid (Lipaphis erysimi) infestation was significantly improved in Brassica juncea through B. juncea-B. fruticulosa introgression. However, linkage drag caused by introgressed chromatin fragments has so far prevented the deployment of this resistance source in commercial cultivars. We investigated the patterns of donor chromatin segment substitutions in the introgression lines (ILs) through genomic in situ hybridization (GISH) coupled with B. juncea chromosome-specific oligonucleotide probes. These allowed identification of large chromosome translocations from B. fruticulosa in the terminal regions of chromosomes A05, B02, B03 and B04 in three founder ILs (AD-64, 101 and 104). Only AD-101 carried an additional translocation at the sub-terminal to intercalary position in both homologues of chromosome A01. We validated these translocations with a reciprocal blast hit analysis using shotgun sequencing of three ILs and species-specific contigs/scaffolds (kb sized) from a de novo assembly of B. fruticulosa. Alien segment substitution on chromosome A05 could not be validated. Current studies also endeavoured to break linkage drag by exposing seeds to a heavy dose (200kR) of gamma radiation. Reduction in the size of introgressed chromatin fragments was observed in many M3 plants. There was a complete loss of the alien chromosome fragment in one instance. A few M3 plants with novel patterns of chromosome segment substitutions displayed improved agronomic performance coupled with resistance to mustard aphid. SNPs in such genomic spaces should aid the development of markers to track introgressed DNA and allow application in plant breeding.

Genetic heterogeneity of disorders with overgrowth and intellectual disability: Experience from a center in North India.

Overgrowth, defined as height and/or OFC ≥ +2SD, characterizes a subset of patients with syndromic intellectual disability (ID). Many of the disorders with overgrowth and ID (OGID) are rare and the full phenotypic and genotypic spectra have not been unraveled. This study was undertaken to characterize the phenotypic and genotypic profile of patients with OGID. Patients with OGID were ascertained from the cohort of patients who underwent cytogenetic microarray (CMA) and/or exome sequencing (ES) at our center over a period of 6 years. Thirty-one subjects (six females) formed the study group with ages between 3.5 months and 13 years. CMA identified pathogenic deletions in two patients. In another 11 patients, a disease causing variant was detected by ES. The spectrum of disorders encompassed aberrations in genes involved in the two main pathways associated with OGID. These were genes involved in epigenetic regulation like NSD1, NFIX, FOXP1, and those in the PI3K-AKT pathway like PTEN, AKT3, TSC2, PPP2R5D. Five novel pathogenic variants were added by this study. NSD1-related Sotos syndrome was the most common disorder, seen in five patients. A causative variant was identified in 61.5% of patients who underwent only ES compared to the low yield of 11.1% in the CMA group. The molecular etiology could be confirmed in 13 subjects with OGID giving a diagnostic yield of 42%. The major burden was formed by autosomal dominant monogenic disorders. Hence, ES maybe a better first-tier genomic test rather than CMA in OGID.

Primary membranous nephropathy in children and adolescents: a single-centre report from South Asia.

BACKGROUND: Unlike adults, primary membranous nephropathy (PMN) comprises only 1-2% of childhood nephrotic syndrome. The clinical behaviour of PMN in children is not explicit and we report upon clinical presentation and outcome. METHODS: This prospective study includes children and adolescents (< 20 years) with biopsy-proven PMN without secondary causes. Anti-PLA2R assessment: before and after completing therapy. OUTCOME: percentage of patients achieving remission. RESULTS: Study cohort included 48 (M:F ratio 1.1:1) patients and median age 17 (IQR 15-18) years, with 35 (72.9%) PLA2R related. Median interval from symptom onset to presentation was 5 months, where median proteinuria, serum albumin and creatinine were 4.9 g/day, 2.1 g/dL and 0.63 mg/dL, respectively. Forty-seven patients received immunosuppressive therapy, with various agents used as first-line therapy: cyclical CYC/GC (53.1%), CNI/GC (21.3%), rituximab (14.9%), prednisolone alone (4.3%), azathioprine (4.3%) and mycophenolate mofetil (2.1%). Median follow-up was 29 (14, 59) months. At 6 months, 11 (24.4%) and 17 (37.7%) had complete remission (CR) or partial remission (PR), while at last follow-up (median 29 months), 20 (45.4%) and 14 (31.8%) had CR and PR respectively. No significant differences in outcome were observed with different agents. A total of 60% patients treated with rituximab as first line/for relapsing disease, and all cases with resistant disease receiving rituximab had CR or PR at last follow-up. PLA2R antibody presence was associated with clinical outcome. CONCLUSIONS: Three-quarters of PMN in children and adolescents is PLA2R related and two-thirds respond to immunosuppressive therapy. Rituximab is a promising agent to manage PMN in children. Anti-PLA2R is associated with clinical outcomes.

Development of a Multiplexed Assay for Detection of Leishmania donovani and Leishmania infantum Protein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a serious and fatal disease caused by the parasites Leishmania infantum and Leishmania donovani The gold standard diagnostic test for VL is the demonstration of parasites or their DNA in spleen, lymph node, or bone marrow aspirates. Serological tests exist but cannot distinguish active VL from either prior exposure to the parasites or previously treated VL disease. Using mass spectroscopy, we have previously identified three L. infantum protein biomarkers (Li-isd1, Li-txn1, and Li-ntf2) in the urine of VL patients and developed a sensitive and specific urine-based antigen detection assay for the diagnosis of VL that occurs in Brazil (where VL is caused by L. infantum). However, unpublished observations from our laboratory at DetectoGen showed that these biomarkers were detected in only 55% to 60% of VL patients from India and Kenya, where the disease is caused by L. donovani Here, we report the discovery and characterization of two new biomarkers of L. donovani (Ld-mao1 and Ld-ppi1) present in the urine of VL patients from these two countries. Capture enzyme-linked immunosorbent assays using specific rabbit IgG and chicken IgY were developed, and the assays had sensitivities of 44.4% and 28.8% for the detection of Ld-mao1 and Ld-ppi1, respectively. In contrast, a multiplexed assay designed to simultaneously detect all five leishmanial biomarkers markedly increased the assay sensitivity to 82.2%. These results validate the utility of leishmanial protein biomarkers found in the urine of VL patients as powerful tools for the development of an accurate diagnostic test for this disease.

Non-Endoplasmic Reticulum-Based Calr (Calreticulin) Can Coordinate Heterocellular Calcium Signaling and Vascular Function.

OBJECTIVE: In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function. APPROACH AND RESULTS: We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr Δ/Δ). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr Δ/Δ mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr Δ/Δ had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER. CONCLUSIONS: Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure.

An Access to Androgen Excess: The Ovary Unregulated.

Hirsutism is excess terminal hair that commonly appears in a male pattern in women. It is associated with hyperandrogenemia. Ferriman-Gallwey scoring system is the most popular scoring system for evaluation, treatment and monitoring the response to therapy. Causes include PCOS, Cushing syndrome, glucocorticoid resistance, drugs, more serious conditions like androgen secreting tumour of ovaries or the adrenal gland, hyperthecosis ovarii and luteoma of pregnancy. We present a case of severe PCOS (Hyperthecosis ovarii) which mimics androgen secreting tumour from ovary in a 30- yr- old lady. This case emphasizes on the spectrum of manifestations that PCOS can come with and the importance of trans vaginal ultrasonography in diagnosing ovarian conditions and its superiority over conventional trans abdominal USG or CT scan.

Hypertension and its severity in children with steroid sensitive nephrotic syndrome during remission.

BACKGROUND AND OBJECTIVE: Hypertension is not a typical feature of steroid sensitive nephrotic syndrome (SSNS) and the presence of persistent hypertension is suggestive of significant renal lesion. There is paucity of data regarding occurrence and severity of hypertension in SSNS in pediatric population during remission and was the main objective of this study. In addition, correlation with factors like family history, BMI, and lipid profile was studied. METHODS: Cross-sectional study conducted at tertiary care center in India including 81 children of infrequent relapsing SSNS between 1 and 10 years in remission and was off steroids. Grading and severity of hypertension were assessed. Statistical analysis was done using SPSS version 21.0. RESULTS: Median age of presentation was 5 years, with male:female ratio of 1.3:1. Out of 81 infrequent relapsing SSNS children, 23.45% (19) had hypertension. Among those children with hypertension (n = 19), 73.68% (14) had positive family history compared to 32.25% (20) in those without hypertension. Positive correlation was found between BP and serum cholesterol and LDL. Of the hypertensive patients, 1 (5.26%) had fundus changes, 2 (10.52%) had features of left ventricular hypertrophy, and 13 (68.42%) had non-nephrotic range proteinuria. CONCLUSION: There is high incidence of hypertension in NS children during remission. Though significant positive correlation was found with positive family history of hypertension and deranged lipid profile highlighting possibility of essential hypertension in them, there is need for close active monitoring and management of hypertension in them as untreated cases may have significant target organ damage.

Dual inhibitors of the human blood-brain barrier drug efflux transporters P-glycoprotein and ABCG2 based on the antiviral azidothymidine.

The brain provides a sanctuary site for HIV due, in part, to poor penetration of antiretroviral agents at the blood-brain barrier. This lack of penetration is partially attributed to drug efflux transporters such as P-glycoprotein (P-gp) and ABCG2. Inhibition of both ABCG2 and P-gp is critical for enhancing drug accumulation into the brain. In this work, we have developed a class of homodimers based on the HIV reverse transcriptase inhibitor azidothymidine (AZT) that effectively inhibits P-gp and ABCG2. These agents block transporter mediated efflux of the P-gp substrate calcein-AM and the ABCG2 substrate mitoxantrone. The homodimers function by interacting with the transporter drug binding sites as demonstrated by competition studies with the photo-affinity agent and P-gp/ABCG2 substrate [125I]iodoarylazidoprazosin. As such, these dual inhibitors of both efflux transporters provide a model for the future development of delivery vehicles for antiretroviral agents to the brain.

Use of PowerPoint presentation as a teaching tool for undergraduate students in the subject of gerodontology.

AIM: Though different teaching learning media have been employed in the instruction of geriatric dentistry, their efficacy has not been adequately evaluated. This study was conducted with the aim to determine the efficacy of a PowerPoint presentation in teaching gerodontology. MATERIALS AND METHODS: This is a prospective follow-up study using a pre- and post-intervention assessment. A pilot study was conducted on the final year students to check the feasibility of the study. A convenience sampling procedure was used. All interns (n = 80) of the Modern Dental College and Research Centre, Indore, India were invited to participate. Interns completed a 24-item questionnaire documenting their current knowledge on gerodontology. One week after a 30 min PowerPoint presentation on gerodontology, the same interns completed the same questionnaire again, providing an indication of the efficacy of the learning tool. Paired t-test and McNemar test were employed for statistical analysis. RESULTS: A statistically significant difference was observed in pre- and post-intervention scores (P < 0.05). CONCLUSION: The results of this study indicated that PowerPoint presentation can be used as an effective tool for improving the knowledge regarding gerodontology.

Principles and application of LIMS in mouse clinics.

Large-scale systemic mouse phenotyping, as performed by mouse clinics for more than a decade, requires thousands of mice from a multitude of different mutant lines to be bred, individually tracked and subjected to phenotyping procedures according to a standardised schedule. All these efforts are typically organised in overlapping projects, running in parallel. In terms of logistics, data capture, data analysis, result visualisation and reporting, new challenges have emerged from such projects. These challenges could hardly be met with traditional methods such as pen & paper colony management, spreadsheet-based data management and manual data analysis. Hence, different Laboratory Information Management Systems (LIMS) have been developed in mouse clinics to facilitate or even enable mouse and data management in the described order of magnitude. This review shows that general principles of LIMS can be empirically deduced from LIMS used by different mouse clinics, although these have evolved differently. Supported by LIMS descriptions and lessons learned from seven mouse clinics, this review also shows that the unique LIMS environment in a particular facility strongly influences strategic LIMS decisions and LIMS development. As a major conclusion, this review states that there is no universal LIMS for the mouse research domain that fits all requirements. Still, empirically deduced general LIMS principles can serve as a master decision support template, which is provided as a hands-on tool for mouse research facilities looking for a LIMS.

Perception and knowledge of dental interns toward interdepartmental coordination for successful prosthodontic treatment: A pilot study.

BACKGROUND: The graduating dentist should be trained in providing treatment utilizing the interdisciplinary approach because of the rapid advancements and increase in patient expectations, which demands for collaboration between the different specialists. PURPOSE: A pilot study was undertaken with an aim to assess the perception and knowledge of dental interns toward interdepartmental coordination for successful prosthodontic treatment. SUBJECTS AND METHODS: Dental interns from two dental colleges in Sangli (India) participated in the study. A 24-item self-administered, structured closed-ended questionnaire was used to collect the data. Four questions assessed the perception and 20 questions assessed the knowledge, which were based on the four domains viz.: General, Endodontics, Orthodontics, and Prosthodontics. They were framed from case scenarios reporting to the Department of Prosthodontics, which required interdepartmental consultation. The questionnaire was validated before its application and reliability were also assessed. The final score for each question was calculated based on the correct responses. Descriptive analysis was calculated using the frequencies, percentages, and mean values by using SPSS 16 software. RESULTS: Among 117 interns who participated in the study, 79.5% reported that they lacked training in an interdisciplinary approach. Approximately, 96% reported that the curriculum should be designed to include interdisciplinary training. Nearly, 88% reported that specialist from different specialty should be posted in one interdisciplinary department. Around 60% reported that they did not have the confidence of treating the patient as a whole. When the overall mean scores were considered, the highest scores were obtained for the general domain (95.3) and the lowest for the prosthodontic domain (83.6). CONCLUSION: The dental interns perceived that they lacked training in interdisciplinary approach, and the curriculum should include interdisciplinary training.

Oncotic Cerebral Aneurysms in a Case of Left Atrial Myxoma, Role of Imaging in Diagnostics and Treatment.

BACKGROUND: Myxomatous cerebral (oncotic) aneurysms following atrial myxoma is a rare neurological complication. CASE REPORT: We report an 11-year- old boy with left atrial myxoma and multiple cerebral oncotic aneurysms. The characteristics of these aneurysms are indefinite and variable. The "Metastasize and Infiltrate" theory may be the key mechanism in the formation of these aneurysms. CONCLUSIONS: Magnetic resonance imaging (MRI), computed tomography (CT) and angiography are useful in the diagnostics while digital subtraction angiography (DSA) is the best option. There are no definite guidelines for therapy of these aneurysms. Resection of cardiac myxomas, chemotherapy, radiotherapy, coil embolization and surgical treatment could be helpful.

Antibiotic prescription pattern among Indian oral healthcare providers: a cross-sectional survey.

OBJECTIVES: Inappropriate prescribing of antibiotics by healthcare professionals is a worldwide concern. The purpose of this study was to determine the pattern of antibiotic prescription among oral healthcare providers in India. METHODS: A one-page questionnaire was sent to 1600 oral healthcare practitioners registered under the Indian Dental Association by using multistage sampling; 552 (34.5%) responded to the survey. The data were analysed using the χ(2) test and multiple logistic regression analysis. RESULTS: Of 552 questionnaire respondents, 405 (73.4%) chose amoxicillin in non-allergic patients, alone [279 (50.5%)] or associated with clavulanic acid [126 (22.8%)]. The average duration of antibiotic therapy was 4.26 ±â€Š1.26 days. The drug of first choice for patients with an allergy to penicillin was erythromycin [242 (43.8%) of respondents]. A majority prescribed antibiotics for irreversible pulpitis and acute apical periodontitis [395 (71.6%)] and necrotic pulp, acute apical periodontitis and no swelling [326 (59.1%)]. Five hundred and ten (92.4%) of the oral healthcare providers overprescribed antibiotics. CONCLUSIONS: Oral healthcare providers in India are overprescribing, which could be a major contributor to the world problem of antimicrobial resistance. As there is overprescription of antibiotics by Indian oral healthcare providers, there is an urgent need to raise public and professional awareness regarding the risks of antibiotic use.

Endodontic management of mesiobuccal-2 canal in four-rooted and five-canalled mandibular third molar.

AIM: A case of unusual Root morphology is presented to demonstrate anatomic variations in mandibular third molar. BACKGROUND: The most common configuration of mandibular third molar is two Roots and three canals; however they may have many different combinations. Endodontic treatment was performed in mandibular third molar having aberrant anatomy. CASE DESCRIPTION: Four Root canal orifices were located with the aid of dental operating microscope (DOM) and three separate Roots were diagnosed with radiographs. Spiral computed tomography (SCT) showed the presence of an extra canal and extra Root, indicating a rare anatomic configuration. Looking for additional canals and Roots are important part of successful endodontics, as the knowledge of their existence enable clinicians to treat a case successfully that otherwise might end in failure. CONCLUSION: The use of DOM and SCT in this case greatly contributed toward making a confirmatory diagnosis and successful endodontic treatment of four-rooted and five-canalled mandibular third molar. CLINICAL SIGNIFICANCE: Variation in Root canal anatomy is very common. Knowledge of these variations is very essential for successful Root canal outcome, inability to do so can lead to missed canals and failures. Hence, thorough knowledge of Root canal anatomy and advances in diagnostic aids are essential.