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Microemulsions are potent drug delivery vehicles as they solubilize water insoluble drugs with poor bioavailability. The main aim of this study was to develop, optimize, and assess a thyme oil microemulsion with inherent antifungal activity for transdermal drug delivery of itraconazole. The optimized thyme oil microemulsion contains thyme oil, Cremophor EL, and water at 5:25:70% v/v respectively. The drug loaded microemulsion was mixed in equal proportion with chitosan for cutaneous application. The droplet size, zeta potential, FTIR spectra, and antifungal activity of the drug free microemulsion and drug loaded microemulsions with/without chitosan were characterized. The ex vivo drug permeation of drug loaded microemulsions with/without chitosan was compared with that of the bulk drug, using the Franz diffusion apparatus with the excised skin from Wistar rats. The droplet size of the drug free microemulsion and drug loaded microemulsions with/without chitosan were 25.53 ± 3.84 nm, 168.6 ± 13.33 nm, and 35.13 ± 0.41 nm respectively, with a polydispersity index between 0.436 and 0.658. The FTIR spectra demonstrated no changes in the structure of the drug in the drug loaded microemulsions with/without chitosan. The in vitro antifungal studies against Candida albicans exhibited similar zones of inhibition between bulk drug and drug loaded microemulsion, while the minimum inhibitory concentration showed a 2-fold decrease with drug loaded microemulsions with/without chitosan compared with the bulk drug. SEM confirmed substantial morphological changes in C. albicans cell membrane. The drug loaded microemulsion with chitosan revealed a slow and sustained release of itraconazole in phosphate buffered saline (pH 7.4) proving it to be an efficient, stable, and sustained topical delivery vehicle.

Antibiotic resistance is one of the most important public health concerns worldwide. Essential oils especially as micro- and nanoemulsion formulations are emerging as potential agents to target resistant pathogens and as drug delivery vehicles. Cinnamon and clove oils were formulated into nanoemulsions using Brij 35 or Tween 20. Based on the physicochemical characteristics and stability, CiB-3, ClB-3, CiT20-5 and ClT20-5 were identified as optimised emulsions and their anti-Staphylococcal properties were determined. Hydrodynamic diameter of the oil droplets was

Many human acid tolerant bacterial and fungal pathogens can be transmitted through the consumption of the contaminated fruit juices. We aim to formulate essential oil nanoemulsions (basil, black seed, turmeric, clove & cinnamon), determine their ability to clear contamination by food borne bacterial pathogens from fruit juices. The antibacterial activity of the optimised formulations was tested in the fruit juices against bacterial pathogens causing gastrointestinal tract infections. The minimum bactericidal concentration (MBC) of clove emulsions ranged from 15.6 to 25 μL mL⁻¹. Cinnamon oil emulsion had an MBC ranging between 15 and 31 μL mL⁻¹. At MBC, cinnamon oil emulsions caused a 6log₁₀ decrease in viable counts by 8 h and maintained the sterility of fruit juices for 7 days at ambient temperature. Thus, clove and cinnamon microemulsions can be used as juice additives to control food borne bacterial pathogens and maintain the bacterial sterility of fruit juices.

Triclosan although banned for the use in many cosmetic products is still used in endodontic therapy for the elimination of microorganisms. At minimum inhibitory concentrations (MIC), triclosan is bacteriostatic and selects resistant clones with prolonged exposure. This study aims to demonstrate the use of eugenol microemulsions as a vehicle to improve the activity of triclosan and prevent the selection of resistant clones. The solubility of triclosan in various oils (cinnamon oil, clove oil, eugenol and peppermint oil) and the release kinetics of triclosan from the microemulsions was determined spectrophotometrically at 281 nm. Microemulsions were formulated by the water titration method. The MIC and minimum bactericidal concentration (MBC) were determined by the broth microdilution method. Triclosan resistant clones were selected by serial passage method. Triclosan has 4 fold difference between its MIC and MBC against S. aureus, E. faecalis and S. mutans isolates. The triclosan loaded microemulsion (5%:25%:70% ratio of oil, Tween 20 & water) (EuTT20-5), containing a final concentration of 3 mg ml−1 of triclosan, eliminated this difference. On repeated passages with increasing concentrations of triclosan, mutant strains of E. faecalis with 8-fold increase in MBC (250 μg ml−1 to 2 mg ml−1) and mutant strains of S. aureus and S. mutans with 8-fold increase in MBC from 125 μg ml−1 to 1 mg ml−1 were observed. Such mutants were not generated in the EuTT20-5 treated cultures. The resistant mutants demonstrated only a 2-fold increase in MBC of EuTT20-5 which is insignificant. Thus, EuTT20-5 can not only improve the efficacy of triclosan, but it can act with equal potency against triclosan resistant isolates. Eugenol loaded triclosan microemulsions can be used instead of conventional biocidal solutions in endodontic therapy where complete elimination of microorganisms from the treatment site is required.

Determining the mutational status of susceptibility genes including RET, VHL, SDHx (SDHB, SDHC, SDHD) among patients with pheochromocytoma/paraganglioma (PCC/PGL) is gaining importance. These genes have not been systematically characterized among patients with PCC/PGL from India. The aim of the work was to screen the most frequently mutated genes among patients with PCC/PGL to determine the frequency and spectrum of mutations seen in this region. Fifty patients with PCC/PGL treated at our tertiary care hospital between January 2010 and June 2012 were screened for mutations in susceptibility genes using an algorithmic approach. Thirty-two percent (16/50) of patients were found to be positive for mutations including mutations among RET (n=4), VHL (n=6), SDHB (n=3), and SDHD (n=3) genes. None of these patients were positive for SDHC mutations. A significant association was found between young patients with bilateral tumors and VHL mutations (p=0.002). Two of the 3 patients with extra-adrenal SDHB associated tumors, had unique mutations, viz., c.436delT (exon 5) and c.788_857del (exon 8), one of which was malignant. High frequency of mutations seen among patients in this study emphasizes the need to consider mutational analysis among Indian patients with PCC/PGL.

Von Hippel-Lindau [VHL] disease, an autosomal dominant hereditary cancer syndrome, is well known for its complex genotype-phenotype correlations. We looked for germline mutations in the VHL gene in an affected multiplex family with Type 1 VHL disease. Real-Time quantitative PCR for deletions and Sanger sequencing of coding regions along with flanking intronic regions were performed in two affected individuals and one related individual. Direct sequencing identified a novel heterozygous single nucleotide base substitution in both the affected members tested, segregating with VHL phenotype in this family. This variant in exon 3, c.473T>A, results in substitution of leucine, a highly conserved acid, to glutamine at position 158 [p.L158Q] and has not been reported thus far as a variant associated with disease causation. Further, this variant was not observed in 50 age and ethnicity matched healthy individuals. Extensive in silico prediction analysis along with molecular dynamics simulation revealed significant deleterious nature of the substitution L158Q on pVHL. The results of this study when collated support the view that the missense variation p.L158Q in the Elongin C binding domain of pVHL may be disease causing.

Neurofibromatosis type 1 is the most common phakomatoses and is inherited in autosomal dominant fashion with complete penetrance. Secondary hypertension is common in these patients due to various causes including adrenal tumors. Pheochromocytoma is a rare catecholamine producing tumor seen in 0.5% to 5% of patients with neurofibromatosis. The combination of pheochromocytoma with neurofibromatosis is rarely reported in the literature. We recently encountered an elderly lady with this combination who successfully underwent adrenalectomy. We report the case for the uncommon occurrence and to highlight the relevant literature review about pheochromocytoma in neurofibromatosis.

Objective: This work aims to improve the oral bioavailability and long-term aqueous stability of curcumin using various edible oil nanoemulsions (NEs). Methods: NEs were optimized using the water titration method. Curcumin was loaded into optimized emulsions, and the physicochemical characteristics were determined. Long-term stability of curcumin in the edible oil NEs was analyzed by determining the droplet size, PDI and curcumin concentrations over 4 mo. Release of curcumin from the NEs was determined using a Franz diffusion apparatus and analysed using 5 mathematical models. Results: The absorbance of curcumin was linear over the concentration range of 1 to 10 μg. ml -1 . The LOD and LOQ ranged from 0.57 to 1.26μg. ml -1 and 1.89 to 4.19μg. ml -1 respectively. All the NEs were monodisperse and had a droplet size less than 150 nm. Long-term emulsion stability shows no change in droplet size and PI (Dunnett's multiple comparisons test with a confidence interval of 95%). Olive oil NE showed significantly low release in gastric fluid (9.28%) with a good release (92.99%) in intestinal fluid and 48% in a body fluid by 8 h. Conclusion: The work highlights the use of olive oil NEs as a delivery vehicle for curcumin with excellent release characteristics and the ability to protect curcumin in an aqueous environment.

Objective: Azithromycin (AZM), an azalide drug is used to treat bacterial infections. It is poorly water-soluble, with low human bioavailability due to partial absorption. This can be improved using a microemulsion drug delivery system using essential oil. Methods: Microemulsion system was prepared with AZM solubilized lemongrass oil ( Cymbopogon citratus ), Tween 20 and water containing 1% (v/v) 10 mmol sodium hydroxide. In vitro drug release was determined using a 14KDa semipermeable dialysis membrane. The kinetics of bacterial killing was done at MIC concentrations, and viable counts were determined hourly for 24 h. Bacterial cell viability was determined by differential staining with acridine orange and ethidium bromide. In vitro toxicity was determined by the MTT assay, while in vivo toxicity was determined in male Wistar rats. Results: The optimized formulation (5:20:75 %) was thermodynamically stable with drug solubility of 366.90 mg/ml and a droplet diameter of 12.4±3.9 nm, which do not show in vivo or in vitro toxicity. In vitro drug release study in simulated body fluids revealed a controlled drug release from microemulsion-based formulation. The MIC was 1μg/ml and 2μg/ml against Staphylococcus aureus and Escherichia coli respectively . In vitro kill kinetics showed>2 log 10 killing by 8 h. Bacterial cell viability assay and scanning electron microscopy analysis further confirmed substantial morphological changes due to alteration in the cell membrane. Conclusion: The reduced droplet size and the inherent antibacterial property of lemongrass oil enhanced the efficacy of the AZM loaded ME system in comparison with the bulk drug, against the bacterial pathogens.

Mutation in VHL gene causes the von Hippel-Lindau (VHL) disease, a dominantly inherited familial cancer syndrome. The VHL mutation pattern includes point mutations, small deletions and large deletions. While most mutations can be identified during sequencing, large deletions often remain unnoticed in initial mutational screening. We evaluated the utility of a previously described real-time quantitative PCR (RQ-PCR) using SYBR Green for detection of larger deletions in the VHL gene and normalized the data using two reference genes with a normal copy number i.e., ZNF80 (3q13.31) and GPR15 (3q12.1). DNA sequencing was also done on all cases included in the study. SJNB-6 cell line demonstrating distal 3p loss was used as a positive control for deletion. Out of 21 individual cases included of VHL disease, 2 cases were found with partial deletion by RQ-PCR, with an exon 1 deletion, while PCR–sequencing identified 5 cases with base pair substitution and 1 with splice site variant which were not picked up by RQ-PCR. RQ-PCR proved to be fast, accurate and sensitive for identifying large deletions and can be incorporated into the routine work-up for detection of large deletions in VHL disease.

Various missense mutations in the VHL gene have been reported among patients with familial bilateral pheochromocytoma. However, the p.Arg82Leu mutation in the VHL gene described here among patients with familial bilateral pheochromocytoma, has never been reported previously in a germline configuration. Interestingly, long-term follow-up of these patients indicated that the mutation might have had little impact on the normal function of the VHL gene, since all of them have remained asymptomatic. We further attempted to correlate this information with the results obtained by in silico analysis of this mutation using SIFT, PhD-SNP SVM profile, MutPred, PolyPhen2, and SNPs&GO prediction tools. To gain, new mechanistic insight into the structural effect, we mapped the mutation on to 3D structure (PDB ID 1LM8). Further, we analyzed the structural level changes in time scale level with respect to native and mutant protein complexes by using 12 ns molecular dynamics simulation method. Though these methods predict the mutation to have a pathogenic potential, it remains to be seen if these patients will eventually develop symptomatic disease.

Objective To describe a family with hereditary paraganglioma due to a disease-causing mutation in the SDHD gene. Methods We present the clinical findings, diagnostic test results, treatment, and genetic test results in a family with hereditary paraganglioma. Results Three siblings with bilateral carotid body tumors presented at different time points and with varied clinical presentations. While the proband, a 20-year-old man, was not hypertensive and had normal urinary metanephrine and normetanephrine levels, his sister and brother had a more severe clinical picture, with hypertension in both and elevated normetanephrine levels in his brother (his brother had pheochromocytoma and 2 intra-abdominal paragangliomas). Mean age at presentation was 24 years. A 4-base pair frameshift mutation, c.337-340delGACT, was detected in exon 4 of the SDHD gene in all 3 patients. Conclusion This is the first report of the c.337340delGACT mutation being associated with hereditary paraganglioma; this report emphasizes the need to screen all at-risk first-degree relatives for the disease-causing SDHD mutation once it has been identified in an affected family member. (Endocr Pract. 2012;18:e106-e110)