Alterations in growth and branching of Neurospora crassa caused by sub-inhibitory concentrations of antifungal agents

Six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in Neurospora crassa. Among the antifungals herein used, the azole agent ketoconazole at 0.5 mu g/ml inhibited radial growth more than fluconazole at 5.0 mu g/ml while amphotericin B at 0.05 mu g/ml was more effective than nystatin at 0.05 mu g/ml. Morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine at the established concentrations. The antifungal agents were more effective on vegetative growth than on conidial germination. Terbinafine markedly reduced growth unit length (GU) by 54.89%, and caused mycelia to become hyperbranched. In all cases, there was a high correlation between hyphal length and number of tips (r > 0.9). All our results showed highly significant differences by ANOVA, (p < 0.001, alpha = 0.05). Considering that the hyphal tip is the main interface between the fungus and its environment/through which enzymes and toxins are secreted and nutrients absorbed, it would not be desirable to obtain a hyperbranched mycelia with inefficient doses of antifungal drugs.

Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21 parts per thousand salinity), as well as potential routes of Na(+) uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase alpha-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell inviginations, respectively. These findings ire discussed regarding the putative movement of Na(+) across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations. (C) 2008 Elsevier Inc. All rights reserved.

Differential adjustment in gill Na(+)/K(+)- and V-ATPase activities and transporter mRNA expression during osmoregulatory acclimation in the cinnamon shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

We evaluate osmotic and chloride (Cl(-)) regulatory capability in the diadromous shrimp Macrobrachium amazonicum, and the accompanying alterations in hemolymph osmolality and [Cl(-)], gill Na(+)/K(+)-ATPase activity, and expression of gill Na(+)/K(+)-ATPase alpha-subunit and V-ATPase B subunit mRNA during salinity (S) acclimation. We also characterize V-ATPase kinetics and the organization of transport-related membrane systems in the gill epithelium. Macrobrachium amazonicum strongly hyper-regulates hemolymph osmolality and [Cl(-)] in freshwater and in salinities up to 25 parts per thousand S. During a 10-day acclimation period to 25 parts per thousand S, hemolymph became isosmotic and hypo-chloremic after 5 days, [Cl(-)] alone remaining hyporegulated thereafter. Gill Na(+)/K(+)-ATPase alpha-subunit mRNA expression increased 6.5 times initial values after 1 h, then decreased to 3 to 4 times initial values by 24 h and to 1.5 times initial values after 10 days at 25 parts per thousand S. This increased expression was accompanied by a sharp decrease at 5 h then recovery of initial Na(+)/K(+)-ATPase activity within 24 h, declining again after 5 days, which suggests transient Cl(-) secretion. V-ATPase B-subunit mRNA expression increased 1.5-fold within 1 h, then reduced sharply to 0.3 times initial values by 5 h, and remained unchanged for the remainder of the 10-day period. V-ATPase activity dropped sharply and was negligible after a 10-day acclimation period to 21 parts per thousand S, revealing a marked downregulation of ion uptake mechanisms. The gill epithelium consists of thick, apical pillar cell flanges, the perikarya of which are coupled to an intralamellar septum. These two cell types respectively exhibit extensive apical evaginations and deep membrane invaginations, both of which are associated with numerous mitochondria, characterizing an ion transporting epithelium. These changes in Na(+)/K(+)- and V-ATPase activities and in mRNA expression during salinity acclimation appear to underpin ion uptake and Cl(-) secretion by the palaemonid shrimp gill.

Structural and Biochemical Correlates of Na(+), K(+)-ATPase Driven Ion Uptake Across the Posterior Gill Epithelium of the True Freshwater Crab, Dilocarcinus pagei (Brachyura, Trichodactylidae)

To better comprehend the structural and biochemical underpinnings of ion uptake across the gills of true freshwater crabs, we performed an ultrastructural, ultracytochemical and morphometric investigation, and kinetically characterized the Na(+), K(+)-ATPase, in posterior gill lamellae of Dilocarcinus pagei. Ultrastructurally, the lamellar epithelia are markedly asymmetrical: the thick, mushroom-shaped, proximal ionocytes contain elongate mitochondria (41% cell volume) associated with numerous (approximate to 14 mu m(2) membrane per mu m(3) cytoplasm), deep invaginations that house the Na(+), K(+)-ATPase, revealed ultracytochemically. Their apical surface is amplified (7.5 mu m(2) mu m(-2)) by stubby evaginations whose bases adjoin mitochondria below the subcuticular space. The apical membrane of the thin, distal ionocytes shows few evaginations (1.6 mu m(2) mu m(-2)), each surrounding a mitochondrion, abundant in the cytoplasm below the subcuticular space; basolateral invaginations and mitochondria are few. Fine basal cytoplasmic bridges project across the hemolymph space, penetrating into the thick ionocytes, suggesting ion movement between the epithelia. Microsomal Na(+), K(+)-ATPase specific activity resembles marine crabs but is approximate to 5-fold less than in species from fluctuating salinities, and freshwater shrimps, suggesting ion loss compensation by strategies other than Na(+) uptake. Enzyme apparent K(+) affinity attains 14-fold that of marine crabs, emphasizing the relevance of elevated K(+) affinity to the conquest of fresh water. Western blotting and biphasic ouabain inhibition disclose two alpha-subunit isoforms comprising distinct functional isoenzymes. While enzyme activity is not synergistically stimulated by NH(4)(+) and K(+), each increases affinity for the other, possibly assuring appropriate intracellular K(+) concentrations. These findings reveal specific structural and biochemical adaptations that may have allowed the establishment of the Brachyura in fresh water. J. Exp. Zool. 313A:508-523, 2010. (C) 2010 Wiley-Liss, Inc.

Biliary and pancreatic dysgenesis in mice harboring a mutation in Pkhd1

Autosomal recessive polycystic kidney disease is a hereditary fibrocystic disease that involves the kidneys and the biliary tract. Mutations in the PKHD1 gene are responsible for typical forms of autosomal recessive polycystic kidney disease. We have generated a mouse model with targeted mutation of Pkbd1 by disrupting exon 4, resulting in a mutant transcript with deletion of 66 codons and expression at similar to 30% of wild-type levels. Pkhd1(del4/d3l4) mice develop intrahepatic bile duct proliferation with progressive cyst formation and associated periportal fibrosis. In addition, these mice exhibit extrahepatic manifestations, including pancreatic cysts, splenomegaly, and common bile duct dilation. The kidneys are unaffected both histologically and functionally. Fibrocystin is expressed in the apical membranes and cilia of bile ducts and distal nephron segments but is absent from the proximal tubule. This pattern is unchanged in orthologous models of autosomal dominant polycystic kidney disease due to mutation in Pkd1 or Pkd2. Mutant fibrocystin in Pkhd1(del4/d3l4) mice also retains this expression pattern. The hypomorphic Pkhd1(del4/d3l4) mouse model provides evidence that reduced functional levels of fibrocystin are sufficient for cystogenesis and fibrosis in the liver and pancreas, but not the kidney, and supports the hypothesis of species-dependent differences in susceptibility of tissues to Pkbdl mutations.

Neural response thresholds in the Nucleus Contour cochlear implant before and after stylet removal

Conclusion. The study shows that there are differences in the measurement of the action potentials with and without the stylet in the Nucleus Freedom Contour Advance that are higher in the apex than in the base of the cochlea. Objectives. To determine if there are differences in the intraoperative impedances and in the neural response telemetry threshold values in the Nucleus Freedom Contour Advance before and after stylet removal. Subjects and methods. This was a prospective clinical study. Intraoperative impedances and neural response telemetry in users of the Freedom Contour Advance Cochlear Implant were measured before and after stylet removal. Results. There was a significant reduction in the impedance values of an average 1.5 k Omega +/- 2.3 in common ground mode and 1.3 k Omega +/- 2.3 for all monopolar modes after the stylet removal (p < 0.001). When analyzing the apical, medium, and basal electrodes, there was a statistically significant reduction in the neural response thresholds after stylet removal only in the apical electrodes (p = 0.001).

Predictive Factors for Positive Surgical Margins and Their Locations After Robot-Assisted Laparoscopic Radical Prostatectomy

Background: Positive surgical margin (PSM) after radical prostatectomy (RP) has been shown to be an independent predictive factor for cancer recurrence. Several investigations have correlated clinical and histopathologic findings with surgical margin status after open RP. However, few studies have addressed the predictive factors for PSM after robot-assisted laparoscopic RP (RARP). Objective: We sought to identify predictive factors for PSMs and their locations after RARP. Design, setting, and participants: We prospectively analyzed 876 consecutive patients who underwent RARP from January 2008 to May 2009. Intervention: All patients underwent RARP performed by a single surgeon with previous experience of > 1500 cases. Measurements: Stepwise logistic regression was used to identify potential predictive factors for PSM. Three logistic regression models were built: (1) one using preoperative variables only, (2) another using all variables (preoperative, intraoperative, and postoperative) combined, and (3) one created to identify potential predictive factors for PSM location. Preoperative variables entered into the models included age, body mass index (BMI), prostate-specific antigen, clinical stage, number of positive cores, percentage of positive cores, and American Urological Association symptom score. Intra-and postoperative variables analyzed were type of nerve sparing, presence of median lobe, percentage of tumor in the surgical specimen, gland size, histopathologic findings, pathologic stage, and pathologic Gleason grade. Results and limitations: In the multivariable analysis including preoperative variables, clinical stage was the only independent predictive factor for PSM, with a higher PSM rate for T3 versus T1c (odds ratio [OR]: 10.7; 95% confidence interval [CI], 2.6-43.8) and for T2 versus T1c (OR: 2.9; 95% CI, 1.9-4.6). Considering pre-, intra-, and postoperative variables combined, percentage of tumor, pathologic stage, and pathologic Gleason score were associated with increased risk of PSM in the univariable analysis (p < 0.001 for all variables). However, in the multivariable analysis, pathologic stage (pT2 vs pT1; OR: 2.9; 95% CI, 1.9-4.6) and percentage of tumor in the surgical specimen (OR: 8.7; 95% CI, 2.2-34.5; p = 0.0022) were the only independent predictive factors for PSM. Finally, BMI was shown to be an independent predictive factor(OR: 1.1; 95% CI, 1.0-1.3; p = 0.0119) for apical PSMs, with increasing BMI predicting higher incidence of apex location. Because most of our patients were referred from other centers, the biopsy technique and the number of cores were not standardized in our series. Conclusions: Clinical stage was the only preoperative variable independently associated with PSM after RARP. Pathologic stage and percentage of tumor in the surgical specimen were identified as independent predictive factors for PSMs when analyzing pre-, intra-, and postoperative variables combined. BMI was shown to be an independent predictive factor for apical PSMs. (C) 2010 European Association of Urology. Published by Elsevier B. V. All rights reserved.

Morphological assessment of dentine and cementum following apicectomy with Zekrya burs and Er:YAG laser associated with direct and indirect Nd:YAG laser irradiation

Objectives. This study aimed to assess the apical surface morphology of maxillary central incisors resected 3.0 mm from the tooth apex using Zekrya burs or Er:YAG laser, with or without subsequent direct Nd:YAG laser irradiation (apical and buccal surfaces) and indirect irradiation (palatal surface). Study design. Forty maxillary central incisors were instrumented and obturated. The roots were divided into 4 groups according to the root resection method (Zekrya bur or Er: YAG laser -1.8 W, 450 mJ, 4 Hz, 113 J/cm(2)) and further surface treatment (none or Nd: YAG laser -2.0 W, 100 mJ, 20 Hz, 124 J/cm(2)). The teeth were prepared for SEM analysis. Scores ranging from 1 to 4 were attributed to cut quality and morphological changes. The data were analyzed by the Kruskal-Wallis test and by Dunn`s test. Results. SEM images showed irregular surfaces on the apical portions resected with Zekrya burs, with smear layer and grooves in the resected dentine and slight gutta-percha displacement and plasticization. On the other hand, apicectomies carried out with Er: YAG laser showed morphological changes compatible with ablated dentine, with rough surfaces and craters. In spite of the presence of plasticized gutta-percha, with the presence of bubbles, an irregular adaptation of the filling material to the root walls was also observed. Direct Nd: YAG laser irradiation of the apical and buccal surfaces of the resected roots resulted in areas of resolidification and fusion in the dentine and cementum, with a vitrified aspect; indirect Nd: YAG laser irradiation of the palatal surfaces yielded a lower number of changes in the cementum, with irregular resolidification areas. Conclusions. There were no differences in terms of cut quality between the use of burs and Er: YAG laser or between the 2 surfaces (apical and buccal) treated with Nd: YAG laser with direct irradiation. However, morphological changes were significantly less frequent on surfaces submitted to indirect irradiation (palatal) when compared with those directly irradiated. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: e77-e82)

Qualitative Analysis of the Removal of the Smear Layer in the Apical Third of Curved Roots: Conventional Irrigation versus Activation Systems

Introduction: The aim of this study was to evaluate the effectiveness of different irrigant agitation techniques on smear layer removal in curved root canals. Methods: Mesiobuccal canals of 62 extracted lower molars with a curvature of 33 degrees were used and instrumented up to Pro Taper F2. The samples were divided into 3 experimental groups according to the final irrigation: conventional irrigation, ultrasonic irrigation, and sonic irrigation by using the Endo Activator system. The control group was composed of 2 specimens without any final irrigation. In all of the experimental groups, 5 mL of 17% ethylenediaminetetraacetic acid was used for 1 minute, and 5 mL of 2.5% NaOCl was used for 30 seconds. The analysis of the apical region was performed via scanning electron microscopy by 3 examiners. The data were submitted to the Kruskal-Wallis and Dunn tests (P<.05). Results: The activation systems removed significantly more smear layer than did conventional irrigation. Conclusions: Sonic and ultrasonic irrigation resulted in better removal of the smear layer in the apical third of curved root canals than did conventional irrigation. (J Endod 2011;37:1268-1271)

Elastic modulus of posts and the risk of root fracture

The definition of an optimal elastic modulus for a post is controversial. This work hypothesized that the influence of the posts` elastic modulus on dentin stress concentration is dependent on the load direction. The objective was to evaluate, using finite element analysis, the maximum principal stress (sigma(max)) on the root, using posts with different elastic modulus submitted to different loading directions. Nine 3D models were built, representing the dentin root, gutta-percha, a conical post and the cortical bone. The softwares used were: MSC.PATRAN2005r2 (preprocessing) and MSC.Marc2005r2 (processing). Load of 100 N was applied, varying the directions (0 degrees, 45 degrees and 90 degrees) in relation to the post`s long axis. The magnitude and direction of the sigma(max) were recorded. At the 45 degrees and 90 degrees loading, the highest values of sigma(max) were recorded for the lowest modulus posts, on the cervical region, with a direction that suggests debonding of the post. For the 0 degrees loading, the highest values of sigma(max) were recorded for higher modulus posts, on the apical region, and the circumferential direction suggests vertical root fracture. The hypothesis was accepted: the effect of the elastic modulus on the magnitude and direction of the sigma(max) generated on the root was dependent on the loading direction.

Influence of dentin on pH of 2% chlorhexidine gel and calcium hydroxide alone or in combination

The aims of endodontic treatment in cases of apical periodontitis are to reduce as much as possible the number of microorganisms inside the root canal system and to inactivate toxins produced by them. Most of the times, these objectives are not achieved solely by chemomechanical preparation, and intracanal dressing may be necessary. In these cases, calcium hydroxide is used as a root canal dressing due to its well-known and recognized antimicrobial activity. Chlorhexidine has a wide spectrum of antimicrobial activity and its association with calcium hydroxide has been recommended in an attempt to amplify antimicrobial effects of calcium hydroxide. It is also known that dentin exerts a buffering effect under wide pH variations, and may be responsible for decreasing the antimicrobial activity of drugs inside the root canal. The objectives of this study were to assess the pH of 2% chlorhexidine gel and calcium hydroxide alone or in combination, as well as the influence of dentin on the pH of these compounds. Dentin powder was obtained from bovine teeth and added as 1.8% to the volume of the medications. All substances were individually stored in plastic flasks, in triplicate. A pH meter was used at five different moments to assess pH in viscous medium: immediately after preparation and after 24 h, and 7, 14, and 21 days. Results were analyzed by paired Student`s t-test. Statistically significant differences were observed in the 2% chlorhexidine gel group alone or associated with calcium hydroxide and added of dentin powder (P < 0.05). Mean pH values indicated the influence of dentin powder because of a significant increase in pH. Calcium hydroxide with propylene glycol as the vehicle always showed high pH, demonstrating that this compound was not affected by the presence of dentin.

Effect of the Combination of Sodium Hypochlorite and Chlorhexidine on Dentinal Permeability and Scanning Electron Microscopy Precipitate Observation

Introduction: This study compared the combined use of sodium hypochlorite (NaOCl) and chlorhexidine (CXH) with citric acid and CXH on dentinal permeability and precipitate formation. Methods: Thirty-four upper anterior teeth were prepared by rotary instrumentation and NaOCl. The root canal surfaces were conditioned for smear layer removal using 15% citric acid solution under ultrasonic activation and a final wash with distilled water. All teeth were dried, and 30 specimens were randomly divided into three equal groups as follows: positive control group (PC), no irrigation; 15% citric acid + 2% CHX group (CA + CHX); and 1% NaOCl + 2% CHX group (NaOCl + CHX). All roots were immersed in a 0.2% Rhodamine B solution for 24 hours. One-millimeter-thick slices from the cementum-enamel junction were scanned at 400 dpi and analyzed using the software ImageLab (LIDO-USP, Sao Paulo, Brazil) for the assessment of leakage in percentage. For scanning electron microscopy analysis, four teeth, irrigated for NaOCl + CHX samples, were split in half, and each third was evaluated at 1,000x and 5,000x (at the precipitate). Results: Using the analysis of variance test followed by the Bonferroni comparison method, no statistical differences between groups were found when analyzed at the cervical and medium thirds. At the apical third, differences between the PC and NaOCl + CHX (p<0.05) and CA + CHX and NaOCl + CHX could be seen (p < 0.05). Conclusion: The combination of 1% NaOCl and 2% CHX solutions results in the formation of a flocculate precipitate that acts as a chemical smear layer reducing the dentinal permeability in the apical third. (J Endod 2010;36:847-850)

Comparison of GPX with or without solvent and hand files in removing filling materials from root canals-An ex vivo study

Objective. The aim of this study was to compare GPX instruments and hand files for gutta-percha removal. Study design. Fifty maxillary central incisors with a single straight canal were instrumented and filled. The teeth were divided into 5 groups of 10 specimens each, according to the gutta-percha removal techniques: group 1: GPX (21-mm-long teeth); group 2: GPX and xylol as solvent (21-mm-long teeth); group 3: GPX (25-mm-long teeth); group 4: GPX and xylol as solvent (25-mm-long teeth); and group 5: hand files and xylol as solvent. The amount of time for gutta-percha removal and the number of fractured instruments were evaluated. Radiographs were taken, and the teeth were grooved longitudinally and split. The area of residual debris was measured using Sigma Scan software. Results. The time for filling material removal was significantly shorter when GPX was used (P < .05). Overall, hand files and solvent produced fewer remnants of filling materials (P < .05). In the GPX 25 mm-long teeth group, the filling material was not removed in the apical third. Conclusions. Under the experimental conditions, the GPX instruments proved to be faster than hand instruments in removing root filling materials; however, hand instruments left a smaller amount of residual filling materials on the canal walls. The GPX instruments did not pull the gutta-percha beyond its tip. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 110: 675-680)

An ex vivo comparison of root canal length determination by three electronic apex locators at positions short of the apical foramen

Objective. The aim of this study was to evaluate the precision of working length determination of 3 electronic apex locators (EALs): Root ZX, RomiApex D-30, and Ipex at 0.0 mm, at the apical foramen (AF), and at 1.0 mm short of the AF. Methodology. Thirty-eight mandibular premolars had their real lengths previously determined. Electronic measurements were determined at 1.0 mm, followed by measurements at 0.0 mm, performed in triplicate. Results. Precision of devices at 1.0 mm and 0.0 mm were: 94.7% and 97.4%, respectively (Root ZX); 78.9% and 97.4% (RomiApex D-30); and 76.3% and 97.4% (Ipex). Although no statistical differences were observed between the EALs at 0.0, at 1.0 mm Root ZX performed significantly better than the others. Conclusion. The EALs had acceptable precision when measuring the working length at the AF. However, when used at levels short of the AF, only Root ZX did not suffer a significant negative effect on precision. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010;110:e57-e61)

Bacterial leakage in obturated root canals-part 2: a comparative histologic and microbiologic analyses

Objective. In this study, presence of dentin infection in root canals, obturated with 4 techniques submitted to the bacterial leakage test, was evaluated using histologic methods. Study design. The canals of palatal roots of 160 molars were instrumented and divided into different groups, according to the obturation technique used (lateral condensation, MicroSeal system, Touch `n Heat + Ultrafil, and Tagger`s hybrid technique) and extent of the remaining obturation material (5 mm and 10 mm). Ten additional roots were used as control samples. The roots were sterilized in ethylene oxide and mounted on a device for evaluation of bacterial leakage using the bacteria Enterococcus faecalis for 120 days. After the leakage test, roots were microscopically analyzed for the presence of dentin infection in the root canals and dentinal tubules. Results. A total of 154 specimens were analyzed using both methodologies in the experimental groups; 50 root canals (32.4%) showed bacterial leakage at the end of the experimental period, and 118 (76.6%) showed the presence of bacteria in the root canals using the histologic criteria. The lateral condensation technique allowed lower penetration of bacteria in the root canals and dentinal tubules, followed by Touch `n Heat + Ultrafil, MicroSeal, and Tagger`s hybrid technique, which allowed significantly greater penetration of bacteria. Root canals with 10 mm of remaining obturation material presented similar bacterial penetration as root canals with 5 mm. Conclusions. Even when an adequate seal of the apical foramen was shown by the absence of turbidity in the bacterial leakage test, E. faecalis dentin infection was present in a high percentage of the root canals after 120 days of root filling exposure to the bacteria. Tagger`s hybrid technique presented greater quantity of bacteria in histologic sections than root canals obturated with the other techniques. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 109: 788-794)