Candida albicans biofilms: comparative analysis of room-temperature and cryofixation for scanning electron microscopy.

Biofilms are frequently related to invasive fungal infections and are reported to be more resistant to antifungal drugs than planktonic cells. The structural complexity of the biofilm as well as the presence of a polymeric extracellular matrix (ECM) is thought to be associated with this resistant behavior. Scanning electron microscopy (SEM) after room temperature glutaraldehyde-based fixation, have been used to study fungal biofilm structure and drug susceptibility but they usually fail to preserve the ECM and, therefore, are not an optimised methodology to understand the complexity of the fungal biofilm. Thus, in this work, we propose a comparative analysis of room-temperature and cryofixation/freeze substitution of Candida albicans biofilms for SEM observation. Our experiments showed that room-temperature fixative protocols using glutaraldehyde and osmium tetroxide prior to alcohol dehydration led to a complete extraction of the polymeric ECM of biofilms. ECM from fixative and alcohol solutions were recovered after all processing steps and these structures were characterised by biochemistry assays, transmission electron microscopy and mass spectrometry. Cryofixation techniques followed by freeze-substitution lead to a great preservation of both ECM structure and C. albicans biofilm cells, allowing the visualisation of a more reliable biofilm structure. These findings reinforce that cryofixation should be the indicated method for SEM sample preparation to study fungal biofilms as it allows the visualisation of the EMC and the exploration of the biofilm structure to its fullest, as its structural/functional role in interaction with host cells, other pathogens and for drug resistance assays.

Determination of impurities in (124)I samples by high resolution gamma spectrometry.

(124)I is a radionuclide used in the diagnosis of tumors. The National Health Agency requires identification and activity measurement of impurities. Using gamma spectrometry with an efficiency calibrated high-purity germanium detector, impurities (125)I and (126)I in an (1)(24)I production sample were identified. Activity ratios of (125)I and (126)I to (124)I were approximately 0.5% and 98%, respectively.

Determination of transit dose profile for a (192)Ir HDR source.

PURPOSE: Several studies have reported methodologies to calculate and correct the transit dose component of the moving radiation source for high dose rate (HDR) brachytherapy planning systems. However, most of these works employ the average source speed, which varies significantly with the measurement technique used, and does not represent a realistic speed profile, therefore, providing an inaccurate dose determination. In this work, the authors quantified the transit dose component of a HDR unit based on the measurement of the instantaneous source speed to produce more accurate dose values. METHODS: The Nucletron microSelectron-HDR Ir-192 source was characterized considering the Task Group 43 (TG-43U1) specifications. The transit dose component was considered through the calculation of the dose distribution using a Monte Carlo particle transport code, MCNP5, for each source position and correcting it by the source speed. The instantaneous source speed measurements were performed in a previous work using two optical fibers connected to a photomultiplier and an oscilloscope. Calculated doses were validated by comparing relative dose profiles with those obtained experimentally using radiochromic films. RESULTS: TG-43U1 source parameters were calculated to validate the Monte Carlo simulations. These agreed with the literature, with differences below 1% for the majority of the points. Calculated dose profiles without transit dose were also validated by comparison with ONCENTRA(®) Brachy v. 3.3 dose values, yielding differences within 1.5%. Dose profiles obtained with MCNP5 corrected using the instantaneous source speed profile showed differences near dwell positions of up to 800% in comparison to values corrected using the average source speed, but they are in good agreement with the experimental data, showing a maximum discrepancy of approximately 3% of the maximum dose. Near a dwell position the transit dose is about 22% of the dwell dose delivered by the source dwelling 1 s and reached 104.0 cGy per irradiation in a hypothetical clinical case studied in this work. CONCLUSIONS: The present work demonstrated that the transit dose correction based on average source speed fails to accurately correct the dose, indicating that the correct speed profile should be considered. The impact on total dose due to the transit dose correction near the dwell positions is significant and should be considered more carefully in treatments with high dose rate, several catheters, multiple dwell positions, small dwell times, and several fractions.

Effects of chronic passive smoking on the regeneration of rat femoral defects filled with hydroxyapatite and stimulated by laser therapy.

Defects associated with bone mass loss are frequently treated by autogenous bone grafting. However, synthetic biomaterials such as calcium phosphate ceramics can substitute autologous grafts as long as they are biocompatible with bone tissue. In addition, low-level laser therapy (LLLT) is used to enhance bone regeneration by stimulating the local microcirculation and increasing the synthesis of collagen by bone cells. However, bone health is fundamental for osseointegration of the graft and bone repair. In this respect, excessive tobacco consumption can compromise expected outcomes because of its deleterious effects on bone metabolism that predispose to the development of osteoporosis. The objective of this study was to evaluate the regeneration of bone defects implanted with biomaterial and stimulated by LLLT in rats submitted to passive cigarette smoking. Porous hydroxyapatite granules were implanted into critical-size defects induced experimentally in the distal epiphysis of the right femur of 20 female Wistar rats submitted to passive smoking for 8 months in a smoking box. The defect site was irradiated with a gallium-arsenide laser at an intensity of 5.0 J/cm2. The animals were divided into four groups: control (non-smoking) rates submitted (G2) or not (G1) to laser irradiation, and smoking rats submitted (G4) or not (G3) to laser irradiation. The animals were sacrificed 8 weeks after biomaterial implantation. The right femurs were removed for photodocumentation, radiographed, and processed for routine histology. The results showed good radiopacity of the implant site and of the hydroxyapatite granules. Histologically, formation of new trabecular bone was observed adjacent to the hydroxyapatite granules in G1 and G2. In G3 and G4, the granules were surrounded mainly by connective tissue. In conclusion, passive smoking compromised bone neoformation in the defects and the LLLT protocol was not adequate to stimulate local osteogenesis.

Study of the prophylactic effect of droperidol, alizapride, propofol and promethazine on spinal morphine-induced pruritus.

BACKGROUND: We have compared the use of alizapride, propofol, droperidol and promethazine for the prevention of spinal morphine-induced pruritus. METHODS: Three hundred ASA I or II women undergoing Caesarean section under spinal anaesthesia, in which morphine 0.2 mg was added to a local anaesthetic, were assigned randomly to receive i.v., in the operating room, just after delivery of the baby, alizapride 100 mg, propofol 20 mg, droperidol 1.25 mg, promethazine 50 mg or saline 2 ml (control group). In the postoperative period, the women were assessed for pruritus (absent, mild, moderate or severe) or other untoward symptoms by blinded observers. We used 95% confidence limits (95% CI) for the cumulative incidence of moderate and severe pruritus to compare the groups, and the NNT and 95% CI to compare droperidol, propofol and alizapride as for their effectiveness in preventing pruritus. For other untoward effects, the chi(2)-test was used, results being considered significant when P<0.05. RESULTS: The droperidol, propofol and alizapride groups had significantly lower incidences of pruritus compared with the control and promethazine groups, while the incidence of pruritus was similar among the patients assigned to the promethazine and control groups. As for the prevention of moderate and severe pruritus, droperidol had the lowest NNT (3.52; 95% CI: 3.37-3.67), followed by propofol (4.61; 95% CI: 4.45-4.77) and alizapride (5.43; 95% CI: 5.27-5.59). As for untoward effects, droperidol and promethazine increased the incidence of somnolence, which seemed more severe with promethazine. Otherwise, there were no differences between the groups. CONCLUSION: Droperidol, propofol and alizapride, in a decreasing order of effectiveness in the doses used in this study, reduced the incidence of pruritus induced by the use of morphine 0.2 mg intrathecally. On the other hand, promethazine 50 mg was shown to be ineffective.