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was suppressed by two orders of magnitude in the presence of tet, and that suppression was reversed when tet was withdrawn.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

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.     

Altered expression of progesterone receptors in testis of infertile men

Progesterone has been implicated in the process of spermatogenesis. This study aimed to investigate the association of progesterone receptor (PR) expression with spermatogenesis in the testis of infertile men. PR mRNA and protein were assessed by in-situ hybridization and immunohistochemistry in testicular biopsies obtained from 18 infertile men. The extent of spermatogenesis was assessed by Johnsen scoring. None of the patients included in the study had Yq microdeletions. PR expression was almost undetectable in all the testicular sections displaying Sertoli cell only (SCO) or arrest at spermatogonia. Weak cytoplasmic expression was observed in biopsies showing arrest at different stages of meiosis. In biopsies displaying spermatogenesis up to the round spermatid stages, PR expression was observed in both nucleus and cytoplasm of different cell types at intensity lower than that detected in normal biopsies. Normal PR expression was observed in biopsies demonstrating hypospermatogenesis. In biopsies showing mixed phenotypes, the tubules with SCO or spermatogonia arrest showed absence of PR expression; normal PR expression was observed in adjacent tubules showing complete spermatogenesis. Semi-quantitative assessment of PR expression and Johnsen scores in the testicular biopsies of infertile men demonstrating different phenotypes indicated a direct relationship between PR expression and extent of spermatogenesis.     

Laryngeal mask airway insertion in children: comparison between rotational, lateral and standard technique

Background:  The purpose of the study was to compare the success and ease of insertion of three techniques of laryngeal mask airway (LMA) insertion; the standard Brain technique, a lateral technique with cuff partially inflated and a rotational technique with cuff partially inflated.
Methods:  One hundred and sixty-eight ASA I and II children aged 6 months to 6 years undergoing short elective surgical procedures lasting 40–60 min were included in the study. A standard anesthesia protocol was followed for all patients. Patients were randomly allocated into one of the three groups i.e. standard (S), rotational (R) and lateral (L). The primary outcome measure of the study was success rate at the first attempt using three techniques of LMA insertion. Secondary outcomes measures studied were overall success rate, time before successful LMA insertion, complications and maneuvers used to relieve airway obstruction.
Results:  Successful insertion at the first attempt was significantly higher in group R (96%) compared with group L (84%) and group S (80%) ( P  =   0.03). Overall success rate (i.e. successful insertion with two attempts) was 100% for group R, 93% for group L and 87% for group S ( P  =   0.03). Time for successful insertion was significantly lower in group R compared with group L and S ( P  <   0.001). The incidence of complications was lower in group R.
Conclusions:  A rotational technique with partially inflated cuff is associated with the highest success rate of insertion and lowest incidence of complications and could be the technique of first choice for LMA insertion in pediatric patients.     

Effect of prophylactic ondansetron on postoperative nausea and vomiting in patients on preoperative steroids undergoing craniotomy for supratentorial tumors

The exact incidence of postoperative nausea and vomiting (PONV) in patients on steroids undergoing neurosurgical procedures is not known. This prospective randomized double-blind study was planned to know the efficacy of prophylactic ondansetron in the prevention of PONV in patients on steroids as compared with placebo. Seventy adult patients of either sex who had received preoperative steroids (dexamethasone) for at least 24 hours and were scheduled to undergo craniotomy for supratentorial tumors were included. Patients were randomly allocated using a randomization chart to 1 of the 2 groups to receive either ondansetron 4 mg (group O) or 0.9% saline (group S) intravenously at the time of dural closure. Numeric Rating Scale score for nausea and pain intensity was recorded preoperatively and till 24 hours postoperatively. The 6-hour postoperative nausea score was significantly lower in group O [median, 0; interquartile range (IQR), 0 to 20] than in group S (median, 20; IQR, 0 to 20) (P<0.05). The incidence of vomiting was lower in group O (23%) than in group S (46%) (P<0.05). The total number of emetic episodes, the number of doses of rescue antiemetics given in the first 6 postoperative hours, and the total number of rescue antiemetics given were significantly lower in group O than in group S (P<0.05). Intravenous administration of 4 mg of ondansetron at the time of dural closure was effective in reducing the incidence of PONV and the rescue antiemetics requirement in patients on preoperative steroids undergoing craniotomy for supratentorial tumors.     

Colour Doppler sonography of flaccid penis in evaluation of erectile dysfunction

BACKGROUND: Erectile dysfunction is a common and potentially treatable problem. Other than psychological, anatomical and metabolic factors, vasculogenic causes also play an important role in erectile dysfunction. Among the various diagnostic tools available for the diagnosis of vasculogenic causes, colour Doppler sonography is noninvasive, simple and promising. METHODS: This preliminary prospective study was conducted on 40 patients with erectile dysfunction, coming from a rural background to a hospital situated in a semi-urban setting. RESULTS: It was found that a cut-off value of 10 cm/second for peak systolic velocity in flaccid penis had the best accuracy among three chosen cut-off values, i.e. 5, 10, 15 cm/second, for detecting arterial insufficiency with sensitivity of 94.1%, specificity of 93.6%, negative predictive value of 98% and positive predictive value of 80%. CONCLUSION: Doppler sonography may be used as a good predictor of clinical response to intracavernosal injection of a vasodilating pharmacological agent.     

Relationship between reduced folate carrier gene polymorphism and non-syndromic cleft lip and palate in Indian population

Objective: Folate metabolism involves absorption, transport, modifications and interconversions of folates. The reduced folate carrier does not participate directly in folate metabolism but plays a major role in intracellular transport of metabolically active 5-methyltetrahydrofolate and maintains the intracellular concentrations of folate. The purpose of this study was to identify the prevalence of reduced folate carrier 1 (RFC1) A80G polymorphism and to further delineate its association with non-syndromic cleft lip and palate (NSCLP) in a south Indian population.

Methods: In the present case-control study, we studied RFC1 gene A80G polymorphism to evaluate its impact on NSCLP risk in south Indian population. Blood samples of 142 cases with NSCLP and 141 controls were collected and genotyped using PCR-RFLP.

Results: The genotype distribution in the control group followed Hardy–Weinberg equilibrium (p?=?0.633). The G allele frequency of cases was 64.8% (184/284) and was significantly lower than that found in the control group 56.4% (160/282). The genotype distributions between NSCLP cases and controls was not significantly different (p?=?0.131). The allelic model significantly increased the risk of NSCLP (G versus A; OR?=?1.40; 95% CI: 1.00–1.97; p?=?0.050). In subgroup analysis, the A80G variant showed significant association for the CLP group in dominant and allelic models.

Conclusions: Altogether, our findings support the hypothesis that RFC1 A80G variant may contribute to NSCLP susceptibility in a south Indian population.