Investigation the effects of silver nanoparticles and gold nanoparticles on expression of bap and csu genes in biofilm formation of Acinetobacter baumannii.

Background and Objectives: Acinetobacter baumannii is one of the main pathogens of the hospital and causes various infections. csu A/BABCDE involved in the initial surface attachment during biofilm formation and bap gene produces specific proteins at the cell surface that play a direct role in formation of biofilm and the infectivity of this bacterium. The aim of this study was to investigate the effect of silver nanoparticles and gold nanoparticles on the expression of bap and csu genes in the Acinetobacter baumannii biofilm formation. Materials and Methods: The susceptibility test was performed to determine the MIC of silver nanoparticles, gold nanoparticles and gold-vancomycin nanoparticles performed by broth dilution method on A. baumannii strains. The ability of biofilms formation in strains treated by MIC of silver nanoparticles and gold-vancomycin nanoparticles were evaluated by microtiter plate method and A. baumannii ATCC19606 used as control. Expression of the csu and bap genes were determinded by measuring the cognate mRNA level by real-time PCR. Results: In present study, gold nanoparticles could not prevent the growth and biofilm formation of A. baumannii strains. The MIC concentration of silver nanoparticles and vancomycin- gold nanoparticles were 6.25 µg/ml and 0.625 µg/ml respectively and MBC concenteration of nanoparticles for 70% of strain was 12.5 µg/ml and 1.25 µg/ml respectively. Real-time PCR and data analysis, determined that the expression of bap, csuC and csuE genes in A. baumannii strains treated with MIC concentration (6.25 µg/ml) of silver nanoparticles decreased compared to control groups. Also, the expression of csuC and csuE genes in strains treated with MIC concentration (0.625 µg/ml) of vancomycin -gold nanoparticles increased, however the expression of bap was decreased compared to the control groups. Conclusion: Due to the inhibitory effect of silver nanoparticles and gold-vancomycin nanoparticles against A. baumannii biofilm formation and genes expression, they can probably be used for prevent of biofilm formation in medical instrument or can be use for treatment of infections with or without antibiotic.

Molecular study and antifungal susceptibility profile of Trichophyton rubrum and Trichophyton mentagrophytes strains isolated from lesions of humans and cattle.

Background and Objectives: Monitoring of contagious diseases is important to advance our knowledge of their epidemiology and to enable more impressive investigation and prevention efforts. This study aimed to examine antifungal drug susceptibility and molecular analysis of clinical isolates of Trichophyton rubrum and Trichophyton mentagrophytes in humans and cattle. Materials and Methods: A total of 400 patients and 500 cattle were evaluated in this study. Dermatophytosis was confirmed in cases by direct microscopy and culture methods. Antifungal drug susceptibility profiles, MIC50, and MIC90 of isolates were determined using the broth microdilution method. Multiplex-PCR, RAPD PCR, and sequencing methods were used for the genetic analysis of virulence genes and the ITS1 and ITS2 regions, respectively. Results: A total of 175 patients and 120 cattle were diagnosed with dermatophytosis. Dermatophytes showed a remarkable rate (30%) of terbinafine resistance. T. mentagrophytes showed lower susceptibility than T. rubrum (MIC50 =16 µg/mL). Strains harboring Mep1, Mep2, and Mep4 genes had the highest frequency among all genotypes. A RAPD-PCR dendrogram divided T. mentagrophytes and T. rubrum strains into three and six groups, respectively. Conclusion: A notable rate of resistance to terbinafine in isolated dermatophytes was reported in this study. Examination of RAPD-PCR results showed that T. rubrum strains had higher genetic diversity than T. mentagrophytes. Genetic monitoring of dermatophytes must be considered an important factor in providing fungal infection prevention and treatment approaches.

Eco-friendly control of licorice aqueous extract to increase quality and resistance to postharvest decay in apple and tangerine fruits.

BACKGROUND: Postharvest diseases in fruits and vegetables are one of the major problems in storing them as a fresh agri-product. This study aimed to investigate the antifungal activity of licorice (Glycyrrhiza glabra) aqueous extract against the Penicillium expansum and the Penicillium digitatum in apple and tangerine fruits as well as their postharvest decay during storage time. METHODS: The minimum inhibitory concentration (MIC) of the molds, and the decay inhibition percentage (%DI) with the P.expansum for apple and P.digitatum for tangerine after treatment with licorice aqueous extract were measured. Additionally, the lesion diameter, titratable acidity (TA), total soluble solids (TSS), pH, and organoleptic properties were determined. RESULTS: The growth of molds was almost inhibited at the concentration of 62.5 mg/mL. The ability of licorice aqueous extract to significantly control and reduce the growth of P. expansum in apple by 60 and 20 % after 7 days and 21 days of storage time was proved, respectively. Furthermore, significant differences in pH and TSS (p < 0.05) were observed in apples. Also, the growth of P. digitatum in the tangerine reduced by 33.3 % after 7 days, while there was no significant difference between the control and treatment groups in pH and TSS for apples, and similarly, there was no significant difference in TA for tangerine samples. CONCLUSIONS: Therefore, the licorice aqueous extract treatment could postpone the blue mold decay in apple fruits and green mold decay in tangerine without any significant effect on fruit quality characteristics. It can be considered as a new eco-friendly control in fruit preservation, while it did not result in any significant adverse effect on  the quality.

Extraction and purification of ß-glucanase from bovine rumen fungus Trichoderma reesei and its effect on performance, carcass characteristics, microbial flora, plasma biochemical parameters, and immunity in a local broiler hybrid Golpayegan-Ross.

The enzyme ß-glucanase was extracted from Trichoderma reesei in bovine rumen fluid samples collected from a slaughterhouse and its effect was investigated in broilers. Data collected was broiler performance, carcass characteristics, duodenum microbial flora, hematological, and immunological parameters. ß-glucanase activity was assayed through spectrometry and was approximately 0.434 IU per gram culture medium. In the current study, endoglucanase enzymes were extracted from Trichoderma reesei. A total of 160 local broilers (Golpayegan-Ross hybrid) were allocated to 4 treatments with 4 replicates per treatment. Over a 49-day experimental period, broilers were fed a basal diet (T1), basal diet plus 20% barley (T2), basal diet with 10 IU extracted ß-glucanase and 20% barley (T3), and basal diet with 10 IU commercial ß-glucanase and 20% barley (T4). The T3 treatment resulted in the greatest body weight gain at the end of experiment (P < 0.01). No significant differences were for feed conversion (FCR; P > 0.05). The highest cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), and LDL cholesterol ratio was observed in the T3 treatment. The highest concentrations of immunoglobulin G1 (IgG1), immunoglobulin G2 (IgG2), and immunoglobulin M1 (IgM1) were observed in the T4 treatment. The T3 treatment resulted in the best response for all measured carcass characteristics. The highest levels of aerobic bacteria, lactobacilli, anaerobic bacteria, and E. coli were associated with the T4, T3, T4, and T1 treatments, respectively. It is concluded that ß-glucanase supplementation can be used to overcome the anti-nutritive effects of water soluble barley non-starch polysaccharides (NSPs) and consequently enhance broiler performance without any adverse effects on humoral immunity parameters.

Biocontrol effect of Kluyveromyces lactis on aflatoxin expression and production in Aspergillus parasiticus.

Aspergillus parasiticus is one of the most common fungi able to produce aflatoxins, which are naturally occurring carcinogenic substances. This study evaluated the effects of the safe yeast, Kluyveromyces lactis, on fungal growth, aflatoxin production and expression of aflR gene in A. parasiticus. Antifungal susceptibility was evaluated by exposing A. parasiticus to different amounts of K. lactis, and aflatoxin production was measured using high-performance liquid chromatography. Expression of the aflR gene was determined by measuring the cognate aflR mRNA level by quantitative real-time reverse-transcription polymerase chain reaction assay. The growth of A. parasiticus was inhibited by 7 days of incubation at 30°C with a minimum population of 1.5 × 105 CFU/ml of K. lactis, which also suppressed expression of the A. parasiticus aflR gene, reducing the total production of aflatoxins by 97.9% and aflatoxins B1, B2, G1 and G2 by 97.8, 98.6, 98 and 94%, respectively. Accordingly, K. lactis could be considered as a potential biocontrol agent against toxigenic molds in food and animal feed.

Comparing Serum and Salivary Levels of Vitamin D in Patients with Recurrent Aphthous Stomatitis and Healthy Individuals.

STATEMENT OF THE PROBLEM: Recurrent aphthous stomatitis (RAS) is the most prevalent ulcerative condition of the oral mucosa. Many studies have emphasized on immunologic factors as the reason of inducing RAS; however, the exact etiologic cause of RAS has not been identified yet. Vitamin D has an endocrine function and regulatory effects on the immune system. It has potential therapeutic effects on autoimmune diseases, psoriasis, and neoplasms. Vitamin D deficiency has been detected in some autoimmune diseases such as rheumatoid arteritis. PURPOSE: The aim of the present study was to compare the serum and salivary levels of vitamin D in patients with RAS and healthy individuals. MATERIALS AND METHOD: In this cross sectional study, patients with RAS, referring to the Department of Oral Medicine, Tabriz Faculty of Dentistry, were evaluated after taking medical history, clinical examinations, and completing an informed consent form. The serum and salivary vitamin D levels were compared between case (n=26) and control (n=26) groups. RESULTS: The mean serum vitamin D levels in the case and control groups were 33.0.7±12.41 and 50.89±9.30 (ng/dL), respectively, with a statistically significant difference (p<0.001). On the other hand, the mean salivary vitamin D levels in the case and control groups were 17.36± 8.01 and 20.79±6.31 (ng/dL), respectively, with no statistically significant difference (p= 0.09). In addition, the correlation between the serum and salivary levels of vitamin D was 56%, being statistically significant (p< 0.001). CONCLUSION: The serum levels of vitamin D in patients with RAS were significantly less than that in healthy individuals; however, there were no significant differences in salivary vitamin D levels between patients with RAS and healthy individuals. In addition, there was a significant and positive correlation between serum and salivary levels of vitamin D in all patients.

Quantification of CDR1 Gene Expression in Fluconazole Resistant Candida Glabrata Strains Using Real-time PCR.

BACKGROUND: The opportunistic fungi, particularly Candida glabrata has been known as main etiologic agents of life-threating infections in some patients. Although fluconazole is the most effective antifungal agent against candidiasis, C. glabrata, fluconazole-resistant strains have been increased recently overexpression or mutations of ATP-binding cassette (ABC) transporter family membrane proteins such as; Cg CDR1, Cg CDR2 are responsible for fluconazole resistance in a large proportion of candidiasis cases. The aim of this study was to evaluate CDR1 gene expression level as one of main mechanism involved in this resistance using. METHODS: Candida glabrata strains were collected from various clinical samples in hospitals of Tehran in 2015 . After validation of all isolates by conventional and molecular methods, the susceptibility analysis to fluconazole of all isolates was performed using CLSI broth microdilution M27-A3 and M27-S4 protocols. Two isolates have been selected based on difference in susceptibility and CDR1-mRNA expression level of isolates was measured by Real-time PCR method. RESULTS: Susceptibility results revealed that 32%, 64% and 4% of strains were susceptible, dose-dependent (DD) and resistant to fluconazole respectively. Furthermore, resistance strain of C. glabrata (MIC≥64 µg/ml) showed overexpression of CDR1 compared with sensitive strain in Real-time PCR analysis. CONCLUSION: Thus, it is necessary to investigate the functions of CgCDR1 genes as a transporter-related gene.

Regulation of ERG3, ERG6, and ERG11 Genes in Antifungal-Resistant isolates of Candida parapsilosis

Background: Candida parapsilosis is one of the five common strains of yeasts involved in invasive candidiasis. The expression analysis of sterol biosynthesis pathway genes, which are associated with resistance, can assist the better understanding of antifungal resistance mechanisms. Method: The antifungal susceptibility of 120 clinical C. parapsilosis isolates was examined. The changes in the gene expression related to resistance were analyzed. Results: Eight strains were resistant to fluconazole (FLC), itraconazole (ITC), and amphotericin B (AMB). The regulation variations included increased mRNA levels of ERG3, ERG6, and ERG11 and decreased mRNA levels of ERG3 and ERG6 in response to FLC. ERG11 mRNA level increases in response to ITC and AMB. Conclusion: The mechanism of resistance to azoles in C. parapsilosis is very similar to C. Albicans. This feature may help to design new treatment strategy for candidiasis.

Effects of Pistacia atlantica subsp. kurdica on Growth and Aflatoxin Production by Aspergillus parasiticus.

BACKGROUND: Aflatoxins are highly toxic secondary metabolites mainly produced by Aspergillus parasiticus. This species can contaminate a wide range of agricultural commodities, including cereals, peanuts, and crops in the field. In recent years, research on medicinal herbs, such as Pistacia atlantica subsp. kurdica, have led to reduced microbial growth, and these herbs also have a particular effect on the production of aflatoxins as carcinogenic compounds. OBJECTIVES: In this study, we to examine P. atlantica subsp. kurdica as a natural compound used to inhibit the growth of A. parasiticus and to act as an anti-mycotoxin. MATERIALS AND METHODS: In vitro antifungal susceptibility testing of P. atlantica subsp. kurdica for A. parasiticus was performed according to CLSI document M38-A2. The rate of aflatoxin production was determined using the HPLC technique after exposure to different concentrations (62.5 - 125 mg/mL) of the gum. The changes in expression levels of the aflR gene were analyzed with a quantitative real-time PCR assay. RESULTS: The results showed that P. atlantica subsp. kurdica can inhibit A. parasiticus growth at a concentration of 125 mg/mL. HPLC results revealed a significant decrease in aflatoxin production with 125 mg/mL of P. atlantica subsp. kurdica, and AFL-B1 production was entirely inhibited. Based on quantitative real-time PCR results, the rate of aflR gene expression was significantly decreased after treatment with P. atlantica subsp. kurdica. CONCLUSIONS: Pistacia atlantica subsp. kurdica has anti-toxic properties in addition to an inhibitory effect on A. parasiticus growth, and is able to decrease aflatoxin production effectively in a dose-dependent manner. Therefore, this herbal extract maybe considered a potential anti-mycotoxin agent in medicine or industrial agriculture.

Investigation the Effects of Lactobacillus acidophilus and Lactobacillus casei on aflR Gene expression in Aspergillus parasiticus by Real Time-PCR.

BACKGROUND: The effect of probiotic bacteria (Lactobacillus acidophilus and L. casei) as safe organisms was examined on fungal growth and aflatoxin gene regulation in Aspergillus parasiticus. METHODS: The fungus was cultured in presence of two different concentrations of L. acidophilus and L. casei in MRS broth medium. Mycelia dry weight is indicated as criteria to evaluate fungal growth. Besides, investigation of aflR gene expression by Real Time PCR was performed for analysis of gene regulatory effects in aflatoxin biosynthetic pathway. RESULTS: Both Lactobacillus strongly inhibited fungal growth in the concentrations of 1. 5×10(2), [Formula: see text] . Expression analysis of aflatoxin genes pathway by real time PCR showed inhibitory effect of L. acidophilus and L. casei on expression of aflR gene. The gene expression revealed to be reduced at the approximate rates of 99. 7% and 98% respectively by L. acidopholus and L. casei in concentrations of [Formula: see text] and more. CONCLUSION: L. acidophilus and L. casei may be used successfully as suitable candidates in controlling of A. parasiticus growth on food and feed as well as reducing of aflatoxin contamination.

Antifungal Susceptibility Analysis of Clinical Isolates of Candida parapsilosis in Iran.

BACKGROUND: Candida parapsilosis is an emergent agent of invasive fungal infections. This yeast is one of the five most widespread yeasts concerned in invasive candidiasis. C. parapsilosis stands out as the second most common yeast species isolated from patients with bloodstream infections especially in neonates with catheter. Recently several reports suggested that its reduced susceptibility to azoles and polyene might become a cause for clinical concern, although C. parapsilosis is not believed to be intensely prone to the development of antifungal resistance. METHODS: In the present report, One hundred and twenty clinical isolates of C. parapsilosis complex were identified and differentiated by using PCR-RFLP analysis. The isolates were then analyzed to determine their susceptibility profile to fluconazole (FLU), itraconazole (ITC) and amphotericin B. The minimum inhibitory concentration (MIC) results were analyzed according to the standard CLSI guide. RESULTS: All of isolates were identified as C. parapsilosis. No C. metapsilosis and C. orthopsilosis strains were found. Evaluation of the antifungal susceptibility profile showed that only three (2.5%) C. parapsilosis were resistant to fluconazole, three (2.5%) C. parapsilosis were resistant to itraconazole and two (1.7%) C. parapsilosis were amphotericin B resistant. CONCLUSION: Profiles in clinical isolates of C. parapsilosis can provide important information for the control of antifungal resistance as well as distribution and susceptibility profiles in populations.

Distinct Toll-like Receptor 3 and 7 Expression in Peripheral Blood Mononuclear Cells From Patients with Chronic Hepatitis C Infection.

BACKGROUND: Hepatitis C virus (HCV) is a major cause of chronic liver disease, with around 130 million infected people worldwide. HCV is recognized by Toll-like receptors (TLRs), which are key mediators of innate immune response. Up on activation of TLRs, anti-viral cytokines and pre-inflammatory are produced. OBJECTIVES: In this study, we compared the expression levels of two members of the TLR family (TLR3 and TLR7) that recognize viral RNA in peripheral blood mononuclear cell (PBMC) of patients with chronic HCV infection and healthy controls. PATIENTS AND METHODS: In this case-control study, blood samples were collected from patients admitted to Blood Transfusion Research Center, Tehran, Iran. PBMC was isolated from blood of chronic HCV patients (n = 25) and age and sex-matched healthy controls (n = 25). RNA was extracted from PBMC and cDNA was synthesized from total RNA templates using reverse transcriptase. The relative level of expression was quantified by real-time PCR using Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene and the results were compared by Pfaffl method. Data were analyzed using non-parametric Wilcoxon test. P < 0.05 was considered significant. RESULTS: In both groups, we had 13 males and 12 females with a mean age of 48.7 ± 16. TLR3 (6.23 ± 0.91 vs. 3.89 ± 0.85, P < 0.001) and TLR7 (1.48 ± 0.82 vs-1.33 ± 1.18, P < 0.001) expressions were significantly lower in patients with chronic HCV infection when compared with healthy controls. CONCLUSIONS: This study suggests that decrease in levels of TLR3 and TLR7 expression is a mechanism that may enable HCV to evade the host innate immune response.

The effect of EFG1 gene silencing on down-regulation of SAP5 gene, by use of RNAi technology.

Efg1 transcription factor is believed to be the main regulator of hyphal formation under many different conditions. In addition, it is responsible for positive regulation of the expression of several hyphal-specific genes. SAP5, which encodes secreted aspartic proteinase, is one of the mentioned genes and is crucial for pathogenicity properties. In the present work we have established the experimental conditions for the use of siRNA in the diploid yeast Candida albicans in order to knock-down the EFG1 gene expression as well as the Efg1-dependent gene, SAP5. The 19-nucleotide siRNA was designed according to cDNA sequence of EFG1 gene in C. albicans and modified-PEG/LiAc method was applied for yeast transfection. To quantify the level of both EFG1 and SAP5 gene expression, the cognate mRNAs were measured in C. albicans by quantitative real-time RT-PCR and data was consequently analyzed by use of REST® software. Images taken by fluorescent microscopy method indicated the effectiveness of transfection. According to REST® software data analysis, expression of EFG1 gene decreased about 2.5-fold using 500 nM of siRNA. A 7-fold decrease in EFG1 gene expression was observed when applying 1 µM of siRNA (P<0.05). Consequently, the expression of SAP5 was significantly down-regulated both in yeast treated with 500 and 1000 nM of siRNA (P<0.05). In conclusion, post-transcriptional gene silencing (PTGS) is likely to be considered as a promising approach to discover new gene targets so as to design fungal-specific antifungal agents, and it is strongly possible that we are taking the right way to battle with C. albicans-associated infections.

Candida species in cutaneous candidiasis patients in the Guilan province in Iran; identified by PCR-RFLP method.

Due to the epidemiological alteration in distribution of Candida species as well as significant increasing trend of either intrinsic or acquired in resistance of some of these fungi, the precise identification of Candida species is necessary for effective antifungal therapy and also for prevention of nosocomial infections. PCR-RFLP method is indicated to be a reliable, rapid and simple technique which is able to differentiate the Candida species. In the present study, we applied this method to evaluate the distribution of Candida species in patients affected with cutaneous candidiasis in the Guilan province. 896 clinical cutaneous samples were collected from different parts of skin and nail of suspected patients referred to clinical centers all over the Guilan province during 24 months. Samples were examined directly with 15% KOH and cultured on fungal specific media. Genomic DNA was extracted and the restriction enzyme Msp1 was applied for polymorphism analysis. Totally, 47 yeast strains were successfully isolated from different clinical samples and identified by conventional as well as PCR-RFLP methods. The results indicated that Candida albicans (36.17%) was the most frequent species followed by C. parapsilosis (25.53%), C. tropicalis (19.14%), C. guilliermondii (14.89%), C. famata (2.12%) and C. krusei (2.12%). Female finger nails were the most common location to be affected by Candida species. In conclusion, PCR-RFLP method was successfully used for recognition of clinical Candida species within the Guilan province and obtained results revealed C. albicans as the predominant causative agent of cutaneous candidiasis. However, distribution of other Candida species did not completely consist with the reported distribution of Candida species in other parts of Iran with different climate to the Guilan province.

Down-regulation of the ALS3 gene as a consequent effect of RNA-mediated silencing of the EFG1 gene in Candida albicans.

BACKGROUND: The most important virulence factor which plays a central role in Candida albicans pathogenesis is the ability of this yeast to alternate between unicellular yeast and filamentous hyphal forms. Efg1 protein is thought to be the main positive regulating transcription factor, which is responsible for regulating hyphal-specific gene expression under most conditions. ALS3 is one of the Efg1-associated genes encoding a multi-functional adhesive polypeptide, which mediates adherence to diverse host substrates. In this study, the EFG1 gene was knocked down by using synthetic siRNA in C. albicans and the regulation in ALS3 as one of the Efg1-dependent genes was investigated. METHOD: The 19-nucleotide siRNA was designed based on cDNA sequence of EFG1 gene in C. albicans. Transfection was performed using modified- plyethylen glycol/LiAc method. To quantify the level of EFG1 and the hyphal-specific ALS3 gene expression, the cognate EFG1 and ALS3 mRNA were measured in C. albicans by quantitative real-time RT-PCR. RESULTS: Fluorescent microscopy pictures indicated that transfection was performed successfully. Also, according to relative expression software tool, expression of EFG1 gene was decreased significantly with 500 nM siRNA as well as 1 µM siRNA (P<0.05). However, more significant down-regulations were observed in the expression of ALS3 in both concentrations of 500 nM and 1 µM siRNA (P<0.05). CONCLUSION: In conclusion, we demonstrated the down-regulation of ALS3 gene as a consequent of applying EFG1-specific siRNA in C. albicans. This may lead us to design anti-fungal-specific agents in order to face with C. albicans-associated infections.

Extracellular production of silver nanoparticles by using three common species of dermatophytes: Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis.

BACKGROUND: To develop a new green approach for biosynthesis of silver nanoparticles, myconanotechnology has been represented as a novel field of study in nanotechnology. In this study, we have reported the extracellular synthesis of highly stable silver nanoparticles using three species of dermatophytes: Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. METHODS: Clinical strains of these species were grown in a liquid medium containing mineral salt and incubated at 25°C for 5-7 days. The cell-free filtrate of each culture was obtained and subjected to synthesize silver nanoparticles in the presence of 1 mM AgNO3. RESULTS: The reduction of Ag+ ions in metal nanoparticles was investigated virtually by tracing the solution color which was switched into reddish-light brown after 72 h. For T. mentagrophytes, a UV-visible spectra demonstrating a strong, quite narrow peak located between 422 and 425 nm was obtained. For M. canis, a fairly wide peak centering at 441 nm and for T. rubrum, a weak spectrum to decipher were observed. According to transmission electron microscopy (TEM) results, fairly uniform, spherical, and small in size with almost less than 50 nm particles were forms in case of T. mentagrophytes. For the other two species, TEM images showed existence of small spherical nanosilvers but not as small as nanoparticles synthesized by T. mentagrophytes. CONCLUSION: We observed that species belong to a single genus of the fungi have variable ability to synthesize silver nanoparticles extracellulary with different efficiency. Furthermore, the extracellular synthesis may make the process simpler and easier for following processes.

RNA-mediated gene silencing in Candida albicans: inhibition of hyphae formation by use of RNAi technology.

The introduction of RNA silencing machinery in fungi has led to the promising application of RNAi methodology to knock down essential vital factor or virulence factor genes in the microorganisms. Efg1p is required for development of a true hyphal growth form which is known to be essential for interactions with human host cells and for the yeast's pathogenesis. In this paper, we describe the development of a system for presenting and studying the RNAi function on the EFG1 gene in C. albicans. The 19-nucleotide siRNA was designed on the basis of the cDNA sequence of the EFG1 gene in C. albicans and transfection was performed by use of a modified-PEG/LiAc method. To investigate EFG1 gene silencing in siRNA-treated cells, the yeasts were grown in human serum; to induce germ tubes a solid medium was used with the serum. Quantitative changes in expression of the EFG1 gene were analyzed by measuring the cognate EFG1 mRNA level by use of a quantitative real-time RT-PCR assay. Compared with the positive control, true hyphae formation was significantly reduced by siRNA at concentrations of 1 µM, 500 nM, and 100 nM (P < 0.05). In addition, siRNA at a concentration of 1 µM was revealed to inhibit expression of the EFG1 gene effectively (P < 0.05). On the basis of the potential of post-transcriptional gene silencing to control the expression of specific genes, these techniques may be regarded as promising means of drug discovery, with applications in biomedicine and functional genomics analysis.

A case of onychomycosis caused by Aspergillus candidus.

Based on epidemiological studies, Aspergillus candidus has been demonstrated as an emerging fungal agent of toenail onychomycosis. Here we report a case of a toenail infection caused by A. candidus in a healthy 60-year-old woman. Based on macroscopic and microscopic characteristics of the culture as well as nucleotide sequencing of 28S region, the causative agent was identified as A. candidus.

A case report of tinea pedis caused by Trichosporon faecale in Iran.

Trichosporon species are known as the causative agents of cutaneous infections and are involved in systemic, localized, as well as disseminated mycoses particularly in immunocompromised patients. Here we report a case of tinea pedis infection caused by Trichosporon faecale in a healthy 29-year-old woman in the north of Iran. Macroscopic and microscopic characteristics using direct examination as well as culture method revealed the causative agent as Trichosporon species. Molecular analysis of the internal transcribed spacer region validated the initial result and indicated that this case of tinea pedis was caused by T. faecale. The patient was recovered after treatment with topical myconazole accompanied with oral fluconazole.

Molecular characterization of a Squalene epoxidase gene in dermatophyte pathogen Trichophyton tonsurans.

BACKGROUND: Trichophyton tonsurans is one of the dermatophyte fungi which invades the skin and hair of human. Several properties of this fungus have been investigated so far. However a few studies were carried out in the field of molecular biology of this fungus. In the present study, we tried to identify the Squalene epoxidase gene which is related to synthesis of ergosterol in this fungus. METHODS: Pairs of 23 and 24 nucleotides primers were designed from highly conserved regions of the similar genes in other fungi. Mentioned primers were utilized in PCR by using isolated genomic DNA of T. tonsurans whereas the PCR fragments were then sequenced. RESULTS AND CONCLUSION: Nucleotides (n = 558) have been sequenced from this new gene which encodes a polypeptide with 186 amino acids. Sequences comparison in gene data banks (NCBI, NIH) for this part of DNA and its deduced amino acid revealed significant homology with members of the eukaryotic Squalene epoxidase.