RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression.

Abasic substitutions within DNA or RNA are tools for evaluating the impact of absent nucleobases. Because of the importance of abasic sites in genetic damage, most research has involved DNA. Little information is available on the impact of abasic substitutions within RNA or on RNA interference (RNAi). Here, we examine the effect of abasic substitutions on RNAi and allele-selective gene silencing. Huntington's disease (HD) and Machado Joseph Disease (MJD) are severe neurological disorders that currently have no cure. HD and MJD are caused by an expansion of CAG repeats within one mRNA allele encoding huntingtin (HTT) and ataxin-3 (ATX-3) proteins. Agents that silence mutant HTT or ATX-3 expression would remove the cause of HD or MJD and provide an option for therapeutic development. We describe flexible syntheses for abasic substitutions and show that abasic RNA duplexes allele-selectively inhibit both mutant HTT and mutant ATX-3. Inhibition involves the RNAi protein argonaute 2, even though the abasic substitution disrupts the catalytic cleavage of RNA target by argonaute 2. Several different abasic duplexes achieve potent and selective inhibition, providing a broad platform for subsequent development. These findings introduce abasic substitutions as a tool for tailoring RNA duplexes for gene silencing.

Allele-selective inhibition of mutant atrophin-1 expression by duplex and single-stranded RNAs.

Dentatorubral-pallidoluysian atrophy (DRPLA) is a progressive neurodegenerative disorder that currently has no curative treatments. DRPLA is caused by an expansion of a CAG trinucleotide repeat region within the protein-encoding sequence of the atrophin-1 (ATN-1) gene. Inhibition of mutant ATN-1 protein expression is one strategy for treating DRPLA, and allele-selective gene silencing agents that block mutant expression over wild-type expression would be lead compounds for therapeutic development. Here we develop an assay for distinguishing mutant from wild-type ATN-1 protein by gel electrophoresis. We use this assay to evaluate duplex RNAs and single-stranded silencing RNAs (ss-siRNAs) for allele-selective inhibition of ATN-1 protein expression. We observed potent and allele-selective inhibition by RNA duplexes that contain mismatched bases relative to the CAG target and have the potential to form miRNA-like complexes. ss-siRNAs that contained mismatches were as selective as mismatch-containing duplexes. We also report allele-selective inhibition by duplex RNAs containing unlocked nucleic acids or abasic substitutions, although selectivities are not as high. Five compounds that showed >8-fold allele selectivity for mutant ATN-1 were also selective for inhibiting the expression of two other trinucleotide repeat disease genes, ataxin-3 (ATXN-3) and huntingtin (HTT). These data demonstrate that the expanded trinucleotide repeat within ATN-1 mRNA is a potential target for compounds designed to achieve allele-selective inhibition of ATN-1 protein, and one agent may allow the targeting of multiple disease genes.

Synthesis, characterization, and evaluation of quaternary ammonium-based polymerizable antimicrobial monomers for prosthodontic applications.

The present study aims to synthesize and characterize two quaternary ammonium (QAM) based monomers such as - dimethyl-hexadecyl-methacryloxyethyl-ammonium iodide (DHMAI) and 2-dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) and assess their cytotoxicity and antimicrobial properties. The study also aims to incorporate the optimized concentration of these monomers as copolymerizing monomers into conventional Polymethyl methacrylate (PMMA) denture base resin and evaluate their suitability for prosthetic applications. DHMAI and DDMAI monomers were synthesized through a Menschutkin reaction and their chemical structure was characterized using FT-IR and 1H-NMR spectroscopy. Cytotoxicity was determined using Methyl Thiazolyl Tetrazolium (MTT) assay whereas antimicrobial activity was assessed using the agar-disc diffusion method. Subsequently, optimized concentrations of DHMAI or DDMAI, based on the cytotoxicity results, were added to conventional PMMA resin. Antimicrobial activity, cytotoxicity, surface hardness, and water sorption of PMMA denture base rein incorporated with DHMAI or DDMAI were evaluated. FT-IR and 1H-NMR results confirmed the structure of monomers and copolymerization of DHMAI and DDMAI with PMMA resin. DHMAI and DDMAI monomers were found to be cytocompatible with mouse fibroblast cells up to a concentration of 5 µg/mL and 20 µg/mL respectively. In addition, incorporation of DHMAI or DDMAI at 5 µg/mL and 20 µg/mL respectively into PMMA denture base material did not affect their cytocompatibility. PMMA denture base resin incorporated with DHMAI or DDMAI significantly reduced the adhesion of microbes. Further, an increase in the surface hardness and a reduction in the water sorption was observed. Hence DHMAI and DDMAI can be considered as potential candidates for imparting antimicrobial activity to polymeric denture base materials.

Comparison of Objective Structured Practical Examination (OSPE) Versus Conventional Pathology Practical Examination Methods Among the Second-Year Medical Students-a Cross-sectional Study.

INTRODUCTION: The medical education in India is moving towards competency-based medical education (CBME) with many cognitive and psychomotor skills needed to be taught and assessed in the various subjects of the undergraduate medical curriculum. In the Pathology subject, psychomotor skills such as haemoglobin estimation, blood grouping, urine examination, liver function test/cerebrospinal fluid interpretation, histopathology and haematology slide interpretation are taught and assessed for many years. Skill assessment by the conventional method, which is followed since many years, is subjective in nature and lack of scope for direct observation of the performance of skills. Objective structured practical examination (OSPE) is one of the methods to minimize the variations in subjectivity, thus enhancing the objectivity. Due to a technicality and labour intensity, it is implemented only in a few medical colleges and universities across India. Because of CBME curriculum on the roll, the assessment of practical skills in medical education needs to be shifted from conventional subjective methods to more objective OSPE methods. MATERIAL AND METHODS: After institutional ethical clearance, the second-year medical students appearing for practical in Pathology were selected for the study. Practical skills of the students were assessed by both the conventional way and OSPE after obtaining the written consent. Among 104 students, 89 students were assessed by both methods. Adequate instructions about the pattern of the examination were given in both assessment methods. For the OSPE group, specific instructions about the role of observer, response stations and method of scoring were given. Practical performance of haemoglobin estimation and blood grouping by slide method was assessed, and scores were compared. Student and faculty perception regarding OSPE was assessed by a prevalidated questionnaire. RESULTS: In the conventional group, the mean score of 6.91 ± 1.08 was obtained, while in OSPE, it was 8.43 ± 1.41. In comparing both, a p value of > 0.001 was obtained, which is found to be significant. Student's perceptions appeared to favour the OSPE format rather than the traditional examination. CONCLUSION: This study showed a significant difference in scores obtained by OSPE in comparison with conventional practical examination. Thus, this information suggests that OSPE format was perceived better by the students, and resulted in a higher average score. Hence, the use of OSPE as a formative assessment tool will help in modifying teaching-learning strategies so that it is beneficial to students and teachers.

Urachal cyst with xanthogranulomatous cystitis: A rare case report.

An urachal cyst is a sinus remaining from the allantois during embryogenesis which is rarely manifested in adults. The urachus is an embryologic remnant which degenerates after the birth. Defective obliteration of the urachus leads to urachal abnormalities. Urachal cyst is a rare pathology in adult women, and this pathology should be considered in the differential diagnosis of acute abdomen. Xanthogranulomatous cystitis (XC) is a benign disease of unknown etiology. The clinical manifestations of these are nonspecific such as lower abdominal pain, umbilical discharge with occasional hematuria. Urachal lesions present with persistent umbilical drainage in infants and newborn. However, in 35% cases, enclosed urachal cyst or infected urachal cyst (abscess) manifests without having umbilical discharge. Computed tomography scan and magnetic resonance imaging are of little help to the identification of these preoperatively. Here, we present a rare case of urachal cyst with XC in 30-year-old female which has produced diagnostic dilemma.

Effectiveness of Laser Fluorescence–Based Device in Detecting the Extent of Re-Mineralisation in Primary Teeth Compared to the Conventional Method: An In vitro Study

Background: Laser fluorescence (LF)–based clinical device DIAGNOdent™ is at present being used to detect caries. Can the same be used to detect therapeutic remineralisation of early white spot lesions? Aims: To explore the feasibility of using LF?based device in monitoring the changes following remineralisation of demineralised primary teeth. Materials and Method: The sample number for the present experimental in vitro study was 10. The LF based device readings were correlated with surface microhardness (SMH) test values to evaluate its efficiency. SMH analysis was performed using a microhardness tester (Tescol?HT1000AD). All the samples were demineralised, followed by remineralisation using fluoride varnish and pH cycling. The data was analysed using the Statistical Package for the Social Sciences (SPSS) version 17.0 (IBM SPSS®) software. Paired t?test was performed to compare laser fluorescence readings and SMH test result values at baseline, after demineralisation, and after remineralisation. Pearson’s correlation was used to compare the relation between the laser fluorescence and SMH test. Results: A good negative correlation was seen between the two methods at the baseline readings even though it was not statistically significant (P = 0.069). A positive correlation between the methods existed following demineralisation which was not significant (P = 0.074). The correlation between the parameters following remineralisation showed a moderate negative correlation but was not significant (P = 0.55). Conclusion: DIAGNOdent™ values at baseline, after demineralisation, and after remineralisation was consistent with SMH values. Thus, DIAGNOdent™ can be explored to provide chairside assistance in identifying remineralisation of white spot lesions.

Unusual presentation of immature teratoma of the neck: A rare case report.

Cervical region teratoma is a rare disease, accounting for 3-5% of all teratomas in the children. Teratomas of the head and neck due to their obscure origin, unpredictable behavior, and often manifest as a clinical surprise. Airway obstruction is the most serious postnatal complication of cervical teratoma. Prenatal diagnosis is crucial for early recognition of the neck masses that could obstruct the airway. We present a case of 4-month-old female child at age of 4 th month with right submandibular region swelling. Computed tomography neck showed ill-defined, multiloculated cystic lesion with enhancing thick septations in the right side of the neck. Excision biopsy revealed Grade I--immature teratoma--cervical region. On 1-year of close follow-up, no evidence of local recurrence or metastasis was seen. Unlike adults, teratomas in children are often congenital and very rarely turn malignant. The treating consultant should be aware of their natural history, clinical features, pathology, and principles of management.

Primary amelanotic melanoma of vulva in a young, lactating female.

Primary amelanotic melanoma of the vulva is extremely rare and it is a unique variant which is difficult to differentiate from other epithelial and nonepithelial malignancies due to absence of melanin pigmentation. It can be easily mistaken for other malignancies both clinically and pathologically. The difficulties in diagnosis and treatment aggravate the poor prognosis. This case highlights the rare case of vulval amelanotic melanoma occurring in a young lactating female.

Synchronous bilateral medullary carcinoma of breast: is it metastasis or second primary?

Bilateral breast cancer is a rare event accounting for 2-5% of all breast malignancies. A second tumor in contralateral breast may be either synchronous or metachronous lesion. Synchronous bilateral invasive ductal carcinoma is known but medullary carcinoma is rare. The etiology of bilateral breast cancer is uncertain and prognosis in these cases once thought to be poor but recent data suggest a similar survival compared to unilateral disease. We report a case of triple negative synchronous bilateral medullary carcinoma in a 38-year-old female who presented with lump in both the breasts for three months. Multidetector computed tomography breast scan revealed bilateral heterogeneously enhancing well-defined lesion in both the breasts. Fine needle aspiration cytology from both the breast lump was suggestive of malignancy. Patient underwent bilateral modified radical mastectomy with axillary clearance in a single sitting. Histopathology showed synchronous bilateral medullary carcinoma of breast with ER, PR and HER- 2/ neu negativity. Patient was treated with chemoradiation and she is on regular follow up for one year without any recurrence or metastasis.

Subcutaneous human dirofilariasis due to dirofilaria repens: report of two cases.

Zoonotic filariasis due to Dirofilaria repens (D. repens) is prevalent in several regions of the world. In view of recent rise of human D. repens infections in Europe, Africa and Asia, it is considered an emerging zoonosis in these continents. Most of the documented cases of human dirofilariasis recorded in India had ocular infections, but very few subcutaneous dirofilariasis have been reported. We hereby report two cases of subcutaneous human dirofilariasis due to D.repens with varied clinical presentations.