The virD operon of Agrobacterium tumefaciens Ti plasmid encodes a DNA-relaxing enzyme.

The virD locus of Agrobacterium tumefaciens Ti plasmid encodes functions necessary for endonucleolytic cleavage of transferred DNA (T-DNA) prior to its transfer to plant cells. For the overproduction of the VIRD proteins in Escherichia coli a tac-virD operon fusion was constructed. A significant increase in the accumulation of VIRD proteins was observed in a lon protease-deficient E. coli host. The presence of an overlapping open reading frame (ORF) upstream of the VIRD1 coding sequence had a negative effect on VIRD1 production. A preparation containing VIRD proteins catalyzes the conversion of supercoiled (form I) DNA to relaxed (form IV) DNA. This activity is similar to that of a DNA topoisomerase. The relaxation activity lacks DNA sequence specificity, requires magnesium ion, and has no requirement for an energy source. Studies with plasmids that had lost defined DNA segments encompassing various virD coding regions showed that VIRD1 is the DNA-relaxing enzyme. In a density gradient centrifugation experiment, the DNA-relaxing activity sedimented as a 21-kDa polypeptide. Earlier studies of Jayaswal et al. [Jayaswal, R., Veluthambi, K., Gelvin, S. & Slightom, J. (1987) (J. Bacteriol. 169, 5035-5045] have shown that in E. coli VIRD2 alone is not sufficient for endonucleolytic cleavage of T-DNA and requires VIRD1 for its activity.     

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was suppressed by two orders of magnitude in the presence of tet, and that suppression was reversed when tet was withdrawn.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.     

Creating patient-centered radiology reports to empower patients undergoing prostate magnetic resonance imaging

IntroductionAs we progress to an era when patient autonomy and shared decision-making are highly valued, there is a need to also have effective patient-centered communication tools. Radiology reports are designed for clinicians and can be very technical and difficult for patients to understand. It is important for patients to understand their magnetic resonance imaging (MRI) report in order to make an informed treatment decision with their physician. Therefore, we aimed to create a patient-centered prostate MRI report to give our patients a better understanding of their clinical condition.MethodsA prototype patient-centered radiology report (PACERR) was created by identifying items to include based on opinions sought from a group of patients undergoing prostate MRI and medical experts. Data was collected in semi-structured interviews using a salient belief question. A prototype PACERR was created in collaboration with human factors engineering and design, medical imaging, biomedical informatics, and cancer patient education groups.ResultsFifteen patients and eight experts from urology, radiation oncology, radiology, and nursing participated in this study. Patients were particularly interested to have a report with laymen terms, concise language, contextualization of values, definitions of medical terms, and next course of action. Everyone believed the report should include the risk of MRI findings actually being cancer in the subsequent biopsy.ConclusionsA prostate MRI PACERR has been developed to communicate the most important findings relevant to decision-making in prostate cancer using patient-oriented design principles. The ability of this tool to improve patient knowledge and communication will be explored.     

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.     

☆Comparison of transcutaneous laryngeal ultrasound with video laryngoscope for assessing the vocal cord mobility in patients undergoing thyroid surgery

ObjectiveWe evaluated the accuracy and feasibility of transcutaneous laryngeal ultrasonography as an alternative to videolaryngoscopy for assessing vocal cord mobility to rule out recurrent laryngeal nerve injury following thyroidectomy.MethodsForty-five adult patients scheduled to undergo elective thyroidectomy under general anesthesia were included. Preoperatively, indirect laryngoscopy and transcutaneous laryngeal ultrasonography was done for assessing vocal cord mobility. Intraoperatively, following induction, patients were intubated using videolaryngoscope. On completion of the surgical procedure, one anesthetist performed videolaryngoscopy so as to record vocal cord mobility while the patients were being extubated in deep plane of anesthesia. Simultaneously another anesthesiologist performed transcutaneous laryngeal ultrasonography.Vocal cord mobility, changes in hemodynamics and total time duration for the two procedures was recorded. Indirect laryngoscopic assessment and flexible fiberoptic laryngoscopy was done on postoperative day 1 and 7 respectively.ResultsPostoperative videolaryngoscopy picked up bilaterally mobile vocal cords in 88.8% cases. Transcutaneous laryngeal ultrasonography could correctly identify 39(86.6%) of these patients, with 1(2.5%) patient being misdiagnosed as having bilaterally immobile vocal cords. Further, videolaryngoscopy identified 5 patients of vocal cord palsy, of which transcutaneous laryngeal ultrasonography correctly identified 3 (60%) patients. Hence, in comparison to videolaryngoscopy, the sensitivity, specificity, positive predictive value, and negative predictive value of transcutaneous laryngeal ultrasonography for assessment of vocal cords was 75%, 95.1%, 60%, and 97.5% respectively.ConclusionIn patients undergoing thyroidectomy, transcutaneous laryngeal ultrasonography can serve as a non-invasive, bedside screening tool for assessing vocal cord palsy postoperatively.     

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.