Cellular apoptosis and cell cycle arrest as potential therapeutic targets for eugenol derivatives in Candida auris.

Candida auris, the youngest Candida species, is known to cause candidiasis and candidemia in humans and has been related to several hospital outbreaks. Moreover, Candida auris infections are largely resistant to the antifungal drugs currently in clinical use, necessitating the development of novel medications and approaches to treat such infections. Following up on our previous studies that demonstrated eugenol tosylate congeners (ETCs) to have antifungal activity, several ETCs (C1-C6) were synthesized to find a lead molecule with the requisite antifungal activity against C. auris. Preliminary tests, including broth microdilution and the MUSE cell viability assay, identified C5 as the most active derivative, with a MIC value of 0.98 g/mL against all strains tested. Cell count and viability assays further validated the fungicidal activity of C5. Apoptotic indicators, such as phosphatidylserine externalization, DNA fragmentation, mitochondrial depolarization, decreased cytochrome c and oxidase activity and cell death confirmed that C5 caused apoptosis in C. auris isolates. The low cytotoxicity of C5 further confirmed the safety of using this derivative in future studies. To support the conclusions drawn in this investigation, additional in vivo experiments demonstrating the antifungal activity of this lead compound in animal models will be needed.

MicroRNA Expression Profile Separates Squamous Cell Carcinoma by Mode of Differentiation.

BACKGROUND: Squamous cell carcinoma (SCC) is a non-melanoma skin cancer with several risk factors including age and sun exposure. The degree of histological differentiation is considered an independent predictor of recurrence, metastasis, and survival. MicroRNAs (miRNAs) are small non-coding RNAs that play an important role in regulating gene expression, culminating in the initiation and progression of multiple tumors. The aim of this study was to determine changes in miRNA expression as a result of the mode of differentiation in SCC. METHODS: We analyzed 29 SCC samples that were separated by mode of differentiation into well (n=4), moderate (n=20) and poor (n=5). Of the 29 samples, five had matched normal tissues, which were used as controls. Total RNA was extracted using the RNeasy FFPE kit, and miRNAs were quantified using Qiagen MiRCURY LNA miRNA PCR Assays. Ten miRNAs (hsa-miR-21, hsa-miR-146b-3p, hsa-miR-155-5p, hsa-miR-451a, hsa-miR-196-5p, hsa-miR-221-5p, hsa-miR-375, hsa-miR-205-5p, hsa-let-7d-5p and hsa-miR-491-5p) that have been previously differentiated in cancer, were quantified. A fold regulation above 1 indicated upregulation and below 1, downregulation. RESULTS: Hierarchical clustering showed that the miRNA expression profile in the moderately differentiated group was similar to the well-differentiated group. The miRNA with the greatest upregulation in the moderate group was hsa-miR-375, while in the well group, hsa-miR-491-5p showed the greatest downregulation. CONCLUSION: In conclusion, this study observed that the well and moderate groups had similar microRNA expression patterns compared to the poorly differentiated group. MicroRNA expression profiling may be used to better understand the factors underpinning mode of differentiation in SCC.

The clinicopathological and microrna expression signature associated with lymphovascular invasion in squamous cell carcinoma: A basic descriptive study.

Background and Aims: Lymphovascular invasion (LVI) is an indicator of lymph node metastasis and poor prognosis in various cancers including squamous cell carcinoma (SCC). Despite being easily resectable and having little potential for LVI; SCC displays aggressive behavior and often results in the death of the patient. With this in mind, it may be useful to investigate the clinical, pathological, and microRNA expression profile associated with LVI in SCC. Methods: We evaluated the histological hallmarks associated with LVI from 16 formalin fixed paraffin embedded (FFPE) tissue samples (10 LVI-, 6 LVI+). We also quantified the expression of 10 microRNAs (hsa-miR-21-5p, hsa-miR-21-3p, hsa-miR-155-5p, hsa-miR-196a-5p, hsa-miR-375, hsa-let-7d-5p, hsa-miR-146b-3p, hsa-miR-221-5p, hsa-miR-205-5p, hsa-miR-491-5p), which have been previously identified to play a role in SCC development, using real time-PCR with the Qiagen miRCURY LNA SYBR Green PCR Kit. Results: We observed a significant upregulation of microRNA-155, microRNA-196a, microRNA-375, and microRNA-221 in cases with lymphovascular invasion. Morphologically, we identified poor differentiation, dysplasia, loss of membrane polarity, high nuclear to cytoplasmic ratio, and the presence of squamous nests as defining features of LVI. Additionally, we found a gender bias and observed a tendency toward lymphatic invasion in lesions presenting around the perineal and abdominal regions. Conclusion: We speculate that this profile may have prognostic significance and could guide the clinician in their treatment protocols for patients matching our genetic, demographic, and morphologic profile.

Perceptions of students on a stand-alone dental materials course in a revised dental curriculum.

BACKGROUND: The Dental Materials (DM) course was introduced as a stand-alone course in 2013, at the University. Prior to that, DM was integrated into clinical courses. OBJECTIVE: To determine the perceptions of the Bachelor of Dental Science (BDS2 to BDS5) students on a stand-alone DM course following curricular amendment. METHODS: This was a cross-sectional study, in which a simple random sampling strategy was used, with forty-six students participating. The study was conducted in 2017. A self-administered, structured, validated questionnaire was used to collect data. The obtained data were summarised and analysed using descriptive and inferential statistics (one-way ANOVA). RESULTS: The study was conducted on Bachelor of Dental Science (BDS2 to BDS5) students. A response rate of 49% from the administered questionnaires was achieved. The overall perception, total mean percentage score of 71.4% was obtained showing a generally positive view on the relevance of the course by students. A total mean percentage score of 74.1% showed the students' view that the DM course was well managed whilst a total mean score of 56.7% expressed the overall view on the reintegration of DM into clinical courses. CONCLUSION: The students felt that DM was relevant and it could continue as a stand-alone course. They perceived that the course was run effectively and managed well, but they had conflicting views on reintegration of the course into clinical modules. Studies with larger sample sizes at other Dental Schools are recommended to determine similarity of results.

The use of real-time polymerase chain reaction and an adenosine deaminase assay for diagnosing pleural tuberculosis.

BACKGROUND: The diagnosis of pleural tuberculosis remains a challenge, because the most widely used conventional diagnostic tools are unable to rapidly detect Mycobacterium tuberculosis in pleural fluid with sufficient sensitivity. OBJECTIVES: The aim of this study was to evaluate the usefulness of an adenosine deaminase assay and real-time polymerase chain reaction (qPCR) in diagnosing pleural tuberculosis. METHODS: One hundred and five consecutive pleural fluid specimens collected between August 2008 and March 2009 were assessed. Among the 105 specimens, 50 (48%) were unconfirmed tuberculosis cases, 21 (20%) were confirmed tuberculosis cases and 34 (32%) were non-tuberculosis cases (controls). Real-time PCR was performed using the Light Cycler Mycobacterium detection kit according to the manufacturer's instructions (Roche Diagnostics). An adenosine deaminase assay was carried out using a commercial colorimetric assay kit as a user-defined method on a Beckman DxC 600 Synchron analyser. RESULTS: The sensitivity of the qPCR was 67% and specificity was 100%. The sensitivity of the adenosine deaminase assay was 80% and specificity was 94%. CONCLUSION: The findings show that the adenosine deaminase assay had higher sensitivity than qPCR. Real-time PCR had 100% specificity, thus a combination of the two methods may be useful for the diagnosis of pleural tuberculosis.

Challenges in the Development of Antifungal Agents Against Candida: Scope of Phytochemical Research.

BACKGROUND: Infections caused by Candida have become a major source of morbidity and mortality. Limited numbers of drugs are available to treat these infections. Phytochemicals can be the major source of antifungal compounds. The aim of this publication was to review the current literature to assess the challenges and scope of phytochemical research in the development of new antifungal drugs. METHODS: Literature describing cellular nature of Candida, the development of drug resistance and target sites for the new drugs were assessed. Publications reporting antifungal activities of crude extracts of plants, their essential oils and identified chemical constituents were also summarised. RESULTS: The results showed that the development of new antifungal agents from natural sources is a complex process due to the cellular nature of Candida and the types of infections caused, such as superficial to life threatening systemic mycosis which necessitate systemic and topical use of drugs. Efficacy of the drugs in the presence of body fluids, normal flora and medical devices can also pose a challenge. Synthetic, semi-synthetic and natural compounds can be screened for their antifungal activities against emerging target sites using new cost effective techniques to increase throughput. Their efficacy, substantivity and site specific desired drug delivery can be enhanced using nanotechnology, hydrogel formulation and bio-adhesive technology. Finally, partnership between academic research laboratories and pharmaceutical industries is also necessary. CONCLUSION: Many challenges are identified in the development of new antifungal drugs, however phytochemicals are still the major source of new antifungal drugs and should be strategically explored.

Synergistic Interactions of Eugenol-tosylate and Its Congeners with Fluconazole against Candida albicans.

We previously reported the antifungal properties of a monoterpene phenol "Eugenol" against different Candida strains and have observed that the addition of methyl group to eugenol drastically increased its antimicrobial potency. Based on the results and the importance of medicinal synthetic chemistry, we synthesized eugenol-tosylate and its congeners (E1-E6) and tested their antifungal activity against different clinical fluconazole (FLC)- susceptible and FLC- resistant C. albicans isolates alone and in combination with FLC by determining fractional inhibitory concentration indices (FICIs) and isobolograms calculated from microdilution assays. Minimum inhibitory concentration (MIC) results confirmed that all the tested C. albicans strains were variably susceptible to the semi-synthetic derivatives E1-E6, with MIC values ranging from 1-62 µg/ml. The test compounds in combination with FLC exhibited either synergy (36%), additive (41%) or indifferent (23%) interactions, however, no antagonistic interactions were observed. The MICs of FLC decreased 2-9 fold when used in combination with the test compounds. Like their precursor eugenol, all the derivatives showed significant impairment of ergosterol biosynthesis in all C. albicans strains coupled with down regulation of the important ergosterol biosynthesis pathway gene-ERG11. The results were further validated by docking studies, which revealed that the inhibitors snugly fitting the active site of the target enzyme, mimicking fluconazole, may well explain their excellent inhibitory activity. Our results suggest that these compounds have a great potential as antifungals, which can be used as chemosensitizing agents with the known antifungal drugs.

Coronal leakage of teeth root-filled with gutta-percha or Resilon root canal filling material.

This study compared the micro-leakage of a root canal filled with Resilon or gutta-percha (GP), utilizing either cold lateral condensation or System B. Four experimental groups were used. Group one was obturated with GP using cold lateral condensation, group two with GP using System B, group three with Resilon using cold lateral condensation, and group four with Resilon using System B. Micro-leakage was tested using a two-chamber bacterial method as well as a dye penetration test. Data was subjected to statistical analysis using an ANOVA. A p-value <0.05 was considered as significant. The bacterial micro-leakage test showed no significant difference between GP and Resilon when using Cold Lateral condensation (p = 0.2695) or System B (p = 0.5602). The dye penetration test also showed no significant difference between GP and Resilon using either the Cold Lateral condensation (p = 0.2713) or the System B techniques (p = 0.0767). The ability of GP and Resilon to seal a root canal is similar.

Clade-related amphotericin B resistance among South African Candida albicans isolates.

Molecular epidemiology revealed 5 distinct clades among clinical isolates of Candida albicans, using DNA fingerprinting with the complex Ca3 probe. Certain clades were found to be highly enriched in particular geographical areas (e.g., clade E in Europe and clade SA in South Africa, whereas clade II is completely absent in the southwest United States). From fingerprinting data, it is concluded that little interclade recombination takes place, and therefore, it would not be unusual to expect clade-specific phenotypic characteristics. The first clade-related phenotypic difference was found with 5-flucytosine resistance being almost exclusively restricted to clade I. When in vitro antifungal susceptibility testing revealed 8.4% of South African oral yeast isolates to be naturally resistant to amphotericin B, it was decided to investigate a possible clade relationship for this relatively high resistance. Thirty-eight amphotericin B-resistant C. albicans isolates were fingerprinted, and a mixed dendrogram was constructed, including previously fingerprinted isolates of known clade affiliation. With the exception of clade III, resistant isolates occurred in all clades (clade I=3; clade II=3; clade NG=3; and clade SA=29), except clade III. However, the higher number of resistant isolates that clustered in clade SA was statistically significant (P