Tetracycline-regulated gene expression following direct gene transfer into mouse skeletal muscle

For most experimental and therapeutic applications of gene transfer, regulation of the timing and level of gene expression is preferable to constitutive gene expression. Among the systems that have been developed for pharmacologically controlled gene expression in mammalian cells, the bacterial tetracycline (tet)-responsive system has the advantage that it is dependent on a drug (tet) that is both highly specific and non-toxic. The tet-responsive system has been previously used to modulate expression of cell cycle regulatory proteins in cultured cells, reporter genes in plants and transgenic mice and reporter genes directly injected into the heart. Here we show that orally or parenterally administered tet regulates expression of tet-responsive plasmids injected directly into mouse skeletal muscle. Reporter gene expression was suppressed by two orders of magnitude in the presence of tet, and that suppression was reversed when tet was withdrawn. These data show that skeletal muscle offers an accessible and well characterized target tissue for tet-controlled expression of genesin vivo, suggesting applications to developmental studies and gene therapy.     

Mechanism of fluconazole resistance in Candida albicans biofilms: phase-specific role of efflux pumps and membrane sterols

Candida albicans biofilms are formed through three distinct developmental phases and are associated with high fluconazole (FLU) resistance. In the present study, we used a set of isogenic Candida strains lacking one or more of the drug efflux pumps Cdr1p, Cdr2p, and Mdr1p to determine their role in FLU resistance of biofilms. Additionally, variation in sterol profile as a possible mechanism of drug resistance was investigated. Our results indicate that parent and mutant strains formed similar biofilms. However, biofilms formed by double and triple mutants were more susceptible to FLU at 6 h (MIC = 64 and 16 microg/ml, respectively) than the wild-type strain (MIC > 256 microg/ml). At later time points (12 and 48 h), all the strains became resistant to this azole (MIC > or = 256 microg/ml), indicating lack of involvement of efflux pumps in resistance at late stages of biofilm formation. Northern blot analyses revealed that Candida biofilms expressed CDR and MDR1 genes in all the developmental phases, while planktonic cells expressed these genes only at the 12- and 48-h time points. Functionality of efflux pumps was assayed by rhodamine (Rh123) efflux assays, which revealed significant differences in Rh123 retention between biofilm and planktonic cells at the early phase (P = 0.0006) but not at later stages (12 and 48 h). Sterol analyses showed that ergosterol levels were significantly decreased (P < 0.001) at intermediate and mature phases, compared to those in early-phase biofilms. These studies suggest that multicomponent, phase-specific mechanisms are operative in antifungal resistance of fungal biofilms.     

Kinetic Evaluation for Removal of an Anionic Diazo Direct Red 28 by Using Phytoremediation Potential of Salvinia molesta Mitchell

Bulletin of Environmental Contamination and Toxicology - The dye removal using phytoremediation has demonstrated its potential to degrade many recalcitrant dyes. The kinetic investigations for...     

Candida biofilm resistance.

Device-related infections in most nosocomial diseases can be traced to the formation of biofilms (microbial communities encased within polysaccharide-rich extracellular matrix) by pathogens on surfaces of these devices. Candida species are the most common fungi isolated from these infections, and biofilms formed by these fungal organisms are associated with drastically enhanced resistance against most antimicrobial agents. This enhanced resistance contributes to the persistence of this fungus despite antifungal therapy. Candida biofilms exhibit enhanced resistance against most antifungal agents, except echinocandins and lipid formulations of AmB. The expression of drug efflux pumps during the early phase of biofilm formation and alterations in membrane sterol composition contribute to resistance of these biofilms against azoles. Metabolic dormancy and ECM do not appear to contribute to resistance, although in a mixed-species biofilm, ECM does retard the diffusion of drugs across biofilm. These multifactorial mechanisms of resistance in fungal biofilms constitute a broad-spectrum defense that is effective against many types of antifungal agents, and represent a common theme present across microbial biofilms.     

NEUROCYSTICERCOSIS IN CHILDREN PRESENTING WITH AFEBRILE SEIZURE: CLINICAL PROFILE,IMAGING AND SERODIAGNOSIS

Neurocysticercosis (NCC) is one of the major causes of childhood seizures in developing countries including India and Latin America. In this study neurological pediatric cases presenting with afebrile seizures were screened for anti-Cysticercus antibodies (IgG) in their sera in order to estimate the possible burden of cysticercal etiology. The study included a total of 61 pediatric afebrile seizure subjects (aged one to 15 years old); there was a male predominance. All the sera were tested using a pre-evaluated commercially procured IgG-ELISA kit (UB-Magiwell Cysticercosis Kit ^™). Anti-Cysticercus antibody in serum was positive in 23 of 61 (37.7%) cases. The majority of cases with a positive ELISA test presented with generalized seizure (52.17%), followed by complex partial seizure (26.08%), and simple partial seizure (21.73%). Headaches were the major complaint (73.91%). Other presentations were vomiting (47.82%), pallor (34.78%), altered sensorium (26.08%), and muscle weakness (13.04%). There was one hemiparesis case diagnosed to be NCC. In this study one child without any significant findings on imaging was also found to be positive by serology. There was a statistically significant association found between the cases with multiple lesions on the brain and the ELISA-positivity (p = 0.017). Overall positivity of the ELISA showed a potential cysticercal etiology. Hence, neurocysticercosis should be suspected in every child presenting with afebrile seizure especially with a radio-imaging supportive diagnosis in tropical developing countries or areas endemic for taeniasis/cysticercosis.     

Can dehydroepiandrostenedione (DHEA) target PRL-3 to prevent colon cancer metastasis?

Colorectal cancer (CRC) is a frequently diagnosed cancer and causing significant mortality in the patients. Metastasis caused by CRC is mainly responsible for this cancer-related deaths. Despite recent advancements in the treatment methods, prognosis remains poor. Therefore, effective treatment strategies need to be designed for successful management of this disease. Dehydroepiandrostenedione (DHEA), a 17-ketosteroid hormone produced by adrenal glands, gonads and including gastrointestinal tract is required for several physiological processes. Deregulation of DHEA levels leads to various disease conditions including cancer. In fact, several experimental studies strongly suggest that DHEA could be used as a chemopreventive agent against colon cancer. Prenlyation of certain membrane proteins such as phosphatase of regenerating liver-3 (PRL-3) is crucial for metastatic progression of colon cancer cells. The ability of DHEA to target prenylation pathway could be utilized to inhibit PRL-3 prenylation for successful prevention of CRC metastases. As DHEA is a widely consumed drug for various ailments, incorporation of DHEA in the treatment regimen may be beneficial to prevent or delay the occurrence of metastasis resulting from CRC.     

Three-dimensional quantitative structure–activity relationship (3D-QSAR) analysis and molecular docking-based combined in silico rational approach to design potent and novel TRPV1 antagonists

The discovery of clinically relevant antagonists of TRPV1 for neuropathy pain therapy has proven to be a challenging task. For better understanding of the molecular interactions of antagonists with TRPV1 receptor, a series of chroman and tetrahydroquinoline ureas were analyzed by k-nearest neighbor molecular field analysis (kNN-MFA) and molecular docking. To elucidate the structural properties required for activity as TRPV1 antagonists, we report here kNN-MFA-based 3D-QSAR model for chroman and tetrahydroquinoline ureas as potent TRPV1 antagonists. Sphere exclusion method was used for dividing the compounds into training (26 compounds) and test (5 compounds) set. Overall model classification accuracy was 81.35 % (q ² = 0.8135, representing internal validation) in training set and 81.44 % (pred_r ² = 0.8144, representing external validation) in test set using stepwise forward as a method of variable selection. The stereo view of molecular rectangular grid field of 3D-QSAR using this approach showed that steric and hydrophobic effects dominantly determine binding affinities. Furthermore, the crystal structure of TRPV1 was obtained from protein data bank (PDB code 2NYJ, resolution 3.20 Å), and docking of 31 TRPV1 antagonists into putative binding sites of the TRPV1 were studies. Molecular docking was employed to explore the binding mode between these compounds and the receptor, as well as help understanding the structure–activity relationship revealed by kNN-MFA. Our QSAR model and molecular docking results corroborate with each other and propose directions for the design of new antagonists with better activity toward TRPV1.