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AIM AND BACKGROUND Azoles as antifungal drugs have been used to treat leishmaniasis for many years. Several evidences suggesting that the primary target of azoles is the heme protein, which co-catalyzes cytochrome P-450-dependent 14α-demethylation of lanosterol. Little is known about the structural changes caused by azoles with atomic force microscopy (AFM) or scanning electron microscopy (SEM). In the current work, several patented antileishmanial agents reviewed (US8809555) (US 0269803 A1) (TW201802093 A). The present study aimed to determine ultrastructural damage in Leishmania major (L.major) induced by the newly synthesized azole. METHODS In this study, we investigated the morphological alterations of the parasite treated with our new synthesized azole namely trans-2-(4-chlorophenyl)-2,3-dihydro-3-(1Himidazol- 1-yl)-4H-1-benzopyran-4-one (IF-2) against L.major promastigotes stage using two high-resolution microscopic techniques: atomic force microscopy and scanning electron microscopy. RESULTS The results showed remarkable topographical and morphological alterations in the cell membrane at promastigote stage of L. major treated with the potent investigated azole (IF-2) ( IC50 values ≤8.9 µg/mL). Both techniques revealed membrane damage and also losing the flagellum in the observed cells. CONCLUSION Our results strongly confirm the Leishmania cell wall as a potent target for the new synthesized azole (IF-2). Accordingly, focus on membrane integrity and glycoconjugates of Leishmania parasite to design new therapeutic agents is recommended.

Trichostrongylus species remain one of the major health challenges in the tropical and summer rainfall regions worldwide. Identification of strongylid species diagnostic methods is vital for obtaining a deep understanding of the epidemiology, population biology, anthelmintic treatment efficacy, and drug resistance in order to design effective parasite control strategies. We evaluated a multiplex RE-PCR for the diagnosis of key Trichostrongylus spp. Genomic DNA amplification of Trichostrongylus colubriformis, Trichostrongylus axei and Trichostrongylus vitrinus was achieved as standard sample using specific primers located in the second internal transcribed spacer (ITSII) of nuclear ribosomal DNA (rDNA). The mentioned method was based on isolation of Trichostrongylus ova from human fecal samples using Willis method, the extraction of ova genomic DNA samples, followed by rDNA ITSII PCR and one-step multiplex RE-PCR using three restriction enzymes of HinfI, DraI, and MseI. The multiplex RE-PCR technique provides a useful tool for discriminating all Trichostrongylus spp., being useful for diagnostic, epidemiological, ecological studies, and control programs. This method is rapid, especially when numerous restriction enzymes are required for species differentiation or identification.

Background The major problem in the management of burn wounds are infections. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major cause of infection in burn wounds. Antibiotic resistant bacteria around the world has become a major therapeutic challenge. Bacteriophages and their lysine are suggested as an antimicrobial alternative agent. Phage display technique is suggested for production of recombinant lysine by Nano carrier technology. The approach of this study was to evaluate the potential of recombinant Nano phage efficacy in MRSA burn wound infection in vivo. Materials and methods The 3rd degree burn wounds were induced in 54 rats and infected with MRSA ATCC 33591 via the topical route in four groups. Burn wound size was measured in 0, 14, 21, 28 days. The efficacy of Nano phage gel was assessed on the basis of percentage collagen deposition, granulation tissue, neovascularization, fibroblastic maturity, re-epithelization, and scar formation in rats following treatment in 14, 21, 28 days. Results The results showed that the percentage of wound size were 3 cm on base line day and the average macroscopic wound healing rates were increased in the prevention groups receiving the recombinant Nano phage gel and natural phage gel, in the treatment groups with secondary infection receiving the recombinant Nano phage gel and the natural phage gel, and in the two control groups respectively. The average microscopic wound healing rates were increased in the prevention groups receiving the recombinant Nano phage gel and natural phage gel, in the treatment groups with secondary infection receiving the recombinant Nano phage gel and the natural phage gel, and in the two control groups respectively. Conclusions In conclusion the recombinant Nano phage gel is efficacy to treat and prevent MRSA burn wound infection.

We describe a microfluidic method for rapid isothermal turbidimetric detection of the DNA of Mycobacterium tuberculosis. Loop-mediated isothermal amplification is accomplished in capillary tubes for amplifying DNA in less than 15 min, and sensitivity and specificity were compared to conventional loop-mediated isothermal amplification (LAMP). The method can detect as little as 1 pg mL−1 DNA in a sample. Results obtained with clinical specimens indicated 90 % sensitivity and 95 % specificity for microfluidic LAMP in comparison to culture methods. No interference occurred due to the presence of nonspecific DNAs. The findings demonstrate the power of the new microfluidic LAMP test for rapid molecular detection of microorganisms even when using bare eyes.

Background: The methicillin-resistant Staphylococcus aureus (MRSA) strain is now the most commonly reported antibiotic-resistant bacterium in clinical settings. Therefore, there is an urgent need to develop novel antibacterial agents to control this pathogen. Bacteriophages are a potential alternative treatment for MRSA infections. Objectives: The objective of this study was the isolation and characterization of MRSA phages from the sewage at a tertiary pediatric hospital. Methods: Two MRSA strains were isolated from patient blood. The isolated strains were confirmed as MRSA through the conventional methods. Phages were then isolated from the sewage at the tertiary pediatric hospital. Lytic activity was determined with a spot test, while the titers of phage lysates were measured using the DLA technique. The morphology was assessed using electron microscopy, and the latent period time and burst size were determined. Results: Electron microscopy showed MRSA phages’ resemblance to members of the family Siphoviridae, serogroups A and F. They exhibited a latent period of 70 minutes and a relatively burst size of 2,400 plaque-forming units (PFU)/infected cell. The titer of the phages was determined to be 3 × 109 PFU/mL using the by DLA technique. Conclusions: In this study, two phages from the family Siphoviridae were isolated and characterized from sewage at a tertiary pediatric hospital; these phages specifically target MRSA. Their presence may be one reason for the transfer of antibiotic resistance or virulence factors, resulting in transduction and the conversion of nonvirulent strains of Staphylococcus to virulent strains. Considering that phages through can be transferred to wards in an air-born manner, it is better to create negative pressure in the sewage pipes at hospitals until the phages can no longer transfer to the wards via aerosols.

The emerging aim of drug delivery requires the bioactive or therapeutic molecule to be protected from degradation and reach its target cell and intracellular location. Target specificity of nanoparticles is a prerequisite to attain the concentration of therapeutic agent required for therapeutic efficacy in the target tissue while minimising adverse effects on other parts of the body. Therefore, there is an urgent need for improvement of more effective drug delivery systems to direct the anticancer drugs to cancer cells, specifically. In the paper, we have described advantages of Lambda bacteriophage over other drug delivery vectors and proposed it as promising drug delivery vehicle. © 2012 Asian Network for Scientific Information.

Firefly luciferase catalyzes the oxidation of luciferin in the presence of ATP, Mg(2+) and molecular oxygen. The bioluminescence color of firefly luciferases is identified by the luciferase structure and assay conditions. Amongst different types of beetles, luciferase from Phrixotrix railroad worm (PhRE) with a unique additional residue (Arg353) naturally emits red bioluminescence color. By insertion of Arg356 in luciferase of Lampyris turkestanicus, corresponding to Arg353 in Phrixotrix hirtus, the color of the emitted light was changed to red. To understand the effect of this position on the bioluminescence color shift, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase. Comparison of mutants with native luciferase shows that mutation brought an increase in the content of secondary structure and globular compactness of (P. pylalis) luciferase. Comparative study of chemical denaturation of native and mutant luciferases by activity measurement, intrinsic and extrinsic fluorescence, circular dichroism, and DSC techniques revealed that insertion of positively charged residues (Arg, Lys) in the flexible loop (352-358) plays a significant role on the stability of (P. pyralis) luciferase and changes the light color to red.

A nanodiagnostic method using nucleic acid sequence-based amplification (NASBA) and gold nanoparticle probes (AuNP probes) was developed for colorimetric detection of Mycobacterium tuberculosis. The primers targeting 16S rRNA were used for the amplification of mycobacterial RNA by the isothermal NASBA process. The amplicons were hybridized with specific gold nanoparticle probes. The RNA–DNA hybrids were colorimetrically detected by the accumulation of gold nanoparticles. Using this method, 10 CFU ml−1 of M. tuberculosis was detected within less than 1 h. Results obtained from the clinical specimens showed 94.7% and 96% sensitivity and specificity, respectively. No interference was encountered in the amplification and detection of M. tuberculosis in the presence of non-target bacteria, confirming the specificity of the method.

This study describes a nanodiagnostic method using thermophilic helicase-dependent isothermal amplification (tHDA) and gold nanoparticle probes for colorimetric detection of Helicobacter pylori DNA. The primers targeting ureC gene were used for the amplification of bacterial DNA by the isothermal tHDA reaction, resulting in the accumulation of DNA amplicons. The amplicons were hybridized with specific gold nanoparticle probes. The hybrids were colorimetrically detected by the assembly of gold nanoparticles. Using this method, we detected as little as 10 CFU mL(-1) of H. pylori within less than 1 h. Results obtained from the gastric biopsy samples showed 92.5% and 95.4% of sensitivity and specificity, respectively, in comparison with culture results, and 100% and 98.8% of sensitivity and specificity, respectively, in comparison with those of the histologic studies. Owing to its ease of operation, this assay significantly reduces the time and cost needed for the molecular diagnosis of H. pylori and has the potential to facilitate early detection of this pathogen.

An enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification (NASBA-ELISA) was developed for molecular detection of Mycobacterium tuberculosis. The primers targeting 16S rRNA were used for the amplification of bacterial RNA by the isothermal digoxigenin (DIG)-labeling NASBA process, resulting in the accumulation of DIG-labeled RNA amplicons. The amplicons were hybridized with a specific biotinylated DNA probe which was non-covalently immobilized on streptavidin-coated microtiter plate. The RNA-DNA hybrids were colorimetrically detected by the addition of an anti-DIG antibody HRP conjugate and 2,2-azino-di-(3-ethylbenzthiazolinsulfonate) substrate. Using this method, as little as 1 x 10(2) CFU ml(-1) of M. tuberculosis was detected within less than 5h. Results obtained from the clinical specimens showed 85.7% and 96% sensitivity and specificity, respectively. No interference was encountered in the amplification and detection of M. tuberculosis in the presence of non-target bacteria, confirming the specificity of the method.

Ionic liquids are recognized as green solvents for carbohydrates dissolution. However, only a limited number of studies have been carried out to investigate their effect on carbohydrate hydrolyzing enzymes. We have investigated the influence of two water miscible ionic liquids on the activity, stability and structure of two related α-amylases from Bacillus amyloliquefaciens and Bacillus lichiniformis. Upon changes in ionic liquids concentrations, both enzymes activity and stability were reduced. Associated thermodynamic and conformational changes were observed using differential scanning calorimetry and fluorescence techniques. Thermal denaturation was accompanied by aggregation in both aqueous buffer and [BMIm][Cl] but [HMIm][Cl] significantly suppressed aggregation.