Bovine sperm cells viability during incubation with or without exogenous DNA

The aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 x 10(6)/ml with or without plasmid pEYFP-NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC-PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.99% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

Placentation in the Amazonian manatee (Trichechus inunguis)

Evidence from several sources supports a close phylogenetic relationship between elephants and sirenians. To explore whether this was reflected in similar placentation, we examined eight delivered placentae from the Amazonian manatee using light microscopy and immunohistochemistry. In addition, the fetal placental circulation was described by scanning electron microscopy of vessel casts. The manatee placenta was zonary and endotheliochorial, like that of the elephant. The interhaemal barrier comprised maternal endothelium, cytotrophoblasts and fetal endothelium. We found columnar trophoblast beneath the chorionic plate and lining lacunae in this region, but there was no trace in the term placenta of haemophagous activity. The gross anatomy of the cord and fetal membranes was consistent with previous descriptions and included a four-chambered allantoic sac, as also found in the elephant and other afrotherians. Connective tissue septae descended from the chorionic plate and carried blood vessels to the labyrinth, where they gave rise to a dense capillary network. This appeared to drain into shorter vessels near the chorionic plate. The maternal vasculature could not be examined in the same detail, but maternal capillaries ran rather straight and roughly parallel to the fetal ones. Overall, there is a close resemblance in placentation between the manatee and the elephant.

PRECLINICAL INVESTIGATION OF THE RETINAL BIOCOMPATIBILITY OF SIX NOVEL VITAL DYES FOR CHROMOVITRECTOMY

Purpose: To investigate the retinal biocompatibility of six novel vital dyes for chromovitrectomy. Methods: An amount of 0.05 mL of 0.5% and 0.05% light green (LG), fast green (FG), Evans blue (EB), brilliant blue (BriB), bromophenol blue (BroB), or indigo carmine (IC) was injected intravitreally in the right eye, whereas in the left eye balanced salt solution was applied for control in rabbits` eyes. Clinical examination, fluorescein angiography, histology with light microscopy, and transmission electron microscopy were performed after 1 and 7 days. Retinal cell layers were evaluated for morphologic alterations and number of cells. The electroretinographic changes were assessed at baseline, 24 hours and 7 days. Results: Fluorescein angiography disclosed hypofluorescent spots only in the 0.5% EB group. Light microscopy and transmission electron microscopy disclosed slight focal morphologic changes in eyes exposed to 0.05% IC, FG, BriB, similar to the control at 1 and 7 days. In the lower dose groups, EB, LG, and BroB caused substantial retinal alterations by light microscopy. At the higher dose, BroB and EB produced diffuse cellular edema and vacuolization within the ganglion cells, bipolar cells, and photoreceptors. FG and IC at 0.5% caused slight retinal alterations similar to balanced salt solution injection. LG at 0.5% caused diffuse vacuolization of bipolar cells after 1 and 7 days. Injection of 0.5% EB caused a significant decrease in neuroretinal cell counts in comparison to control eyes in the 7-day examination (P < 0.05). Electroretinography revealed intermittent prolonged latency and decreased amplitude in eyes injected with 0.5% EB, LG, BriB, and BroB, while at the lower dose, only LG and EB induced few functional changes. Conclusion: The progressive order of retinal biocompatibility, from safest to most toxic, was IC, FG, BriB, BroB, LG, EB.

The neural histogenetic origin of the oral granular cell tumor: An immunohistochemical evidence

Aims: Granular cell tumor (GCT) is a rare neoplasm that can appear in any site of the body, but most are located intraorally. Its histogenetic origin remains unclear. This report analyzes the immunoprofile of 15 cases of granular cell tumors, occurring in 13 women and 2 men and the lesions were located on the tongue or upper lip. Patient age ranged from 7 to 52. Methods: The patients demographic data and the cytological and architectural features of the lesions were analyzed in oral GCTs (n = 15). The lesions were also submitted to a panel of immunohistochemical stains with antibodies against S-100, p75, NSE, CD-68, Ki-67, Synaptofisin, HHF-35, SMA, EMA, Chromogranin, Progesterone, Androgen and Estrogen. Results: Among the fifteen cases analyzed, the most common location was the tongue (84.6%). Histologically, the tumors exhibited cellular proliferation composed mainly by polygonal cells presenting an abundant granular eosinophilic cytoplasm. The nuclei were central, and the cell membranes were moderately clear. No mitotic figures were observed. The immunohistochemical analysis showed positivity in all cases for S-100, p75, NSE and CD-68, and no immunoreactivity for Ki-67, Synaptofisin, HHF-35, SMA, EMA, Chromogranin, Progesterone, Androgen and Estrogen. Conclusion: The immunoprofile of granular cell tumors showed nerve sheath differentiation - lending support to their neural origin - and helping to establish a differential diagnosis between this lesion and other oral granular cell tumors, whether benign or malignant.

Treatment of infrabony defects with or without enamel matrix proteins: A 24-month follow-up randomized pilot study

Objective: To evaluate a comparison of open-flap debridement (OFD) with or without the use of enamel matrix proteins (EMP) for the treatment of infrabony defects. Method and Materials: Ten volunteers (38 infrabony defects) were randomized to receive OFD + EMP (test site) and OFD (control site). Clinical outcomes included mean changes in Plaque Index, Gingival Index, probing pocket depth (PPD), relative attachment level (RAL), gingival recession, width of keratinized tissue, and dental mobility at baseline and at 24 months. Results: A significant reduction of 4.21 +/- 0.97 mm was observed in PPD for the OFD + EMP group (from 6.30 +/- 0.99 mm to 2.09 +/- 0.97 mm) and of 3.28 +/- 1.23 mm for the OFD group (from 6.13 +/- 0.88 mm to 2.85 +/- 1.42 mm) (P < .001). The reduction in PPD was statistically significantly greater for OFD + EMP compared to OFD (P = .03). The mean RAL decreased from 13.26 +/- 1.88 mm to 7.57 +/- 2.05 mm for the OFD + EMP group (a gain of 5.69 +/- 1.96 mm) and from 13.37 +/- 1.71 mm to 8.13 +/- 1.34 min (P < .001) for the OFD group (a gain of 5.24 +/- 1.55 mm). Gingival recession was higher it) the OFD + EMP group than in the OFD group. The mean keratinized tissue significantly decreased from 4.41 +/- 1.39 mm to 3.63 +/- 1.54 mm for OFD flap group (P < .01). Conclusion: Both treatment modalities were efficient in improving RAL and PPD. Within groups, there was a significant reduction in keratinized tissue for OFD and a significant postoperative recession for the OFD + EMP group. Infrabony defects treated with OFD + EMP showed significantly more PPD reduction when compared to OFD. (Quintessence Int 2010;41:125-134)

Can the durability of one-step self-etch adhesives be improved by double application or by an extra layer of hydrophobic resin?

Objectives: This study evaluated the immediate and 6-month resin-dentin mu-bond strength (mu TBS) of one-step self-etch systems (Adper Prompt L-Pop [AD] 3M ESPE; Xeno III [XE] Dentsply De Trey; iBond [iB] Heraeus Kulzer) under different application modes. Materials and methods: Dentin oclusal surfaces were exposed by grinding with 600-grit SiC paper. The adhesives were applied according to the manufacturer`s directions [MD], or with double application of the adhesive layer [DA] or following the manufacturer`s directions plus a hydrophobic resin layer coating [HL]. After applying the adhesive resins, composite crowns were built up incrementally. After 24-h water storage, the specimens were serially sectioned in ""x"" and ""y"" directions to obtain bonded sticks of about 0.8 mm 2 to be tested immediately [IM] or after 6 months of water storage [6M] at a crosshead speed of 0.5 mm/min. The data from each adhesive was analyzed by a two-way repeated measures ANOVA (mode of application vs. storage time) and Tukey`s test (alpha = 0.05). Results: The adhesives performed differently according to the application mode. The DA and HL either improved the immediate performance of the adhesive or did not differ from the MD. The resin-dentin bond strength values observed after 6 months were higher when a hydrophobic resin coat was used than compared to those values observed under the manufacturer`s directions. Conclusions: The double application of one-step self-etch system can be safety performed however the application of an additional hydrophobic resin layer can improve the immediate resin-dentin bonds and reduce the degradation of resin bonds over time. (c) 2008 Elsevier Ltd. All rights reserved.

Microshear Bond Strength of Self-etching Systems Associated with a Hydrophobic Resin Layer

Purpose: To evaluate in vitro the microshear bond strength of adhesive systems applied to dentin according to manufacturers` instructions, associated or not with a hydrophobic layer of unfilled resin. Materials and Methods: Six self-etching adhesives (Clearfil SE Bond, Kuraray Medical; AdheSE, lvoclar Vivadent; Xeno III, Dentsply; I Bond, Heraeus-Kulzer; Bond Force, Tokuyama; Futurabond DC, Voco) were tested. The labial dentin of sixty bovine incisors was exposed, and the teeth were divided into two groups according to the application or not of an extra hydrophobic resin layer (Scotchbond Multi Purpose Plus, bottle 3). Six composite cylinders (Filtek Z250, 3M ESPE) were built up on each treated surface. Specimens were stored in distilled water at 37 C for 24 h and then subjected to the microshear bond strength test in a universal testing machine at a crosshead speed of 0.5 mm/min. Microshear bond strength values were analyzed by 2-way ANOVA and Tukey`s post-hoc test. Failure mode was determined using a stereomicroscope under 20X magnification. Results: The application of the hydrophobic resin layer did not affect bond strength, except for AdheSE. However, the bond strengths with the hydrophobic layer were similar among the six tested systems (Clearfil: 17.1 +/- 7.9; AdheSE: 14.5 +/- 7.1; Xeno III: 12.8 +/- 7.7; I Bond: 9.5 +/- 5.8; Bond Force: 17.5 +/- 4.1; Futurabond: 7.7 +/- 2.3). When used as recommended by the manufacturers, Bond Force presented statistically higher bond strength than AdheSE and I Bond (p < 0.05) (Clearfil 10.4 +/- 4.9; AdheSE 1.6 +/- 1.6; Xeno III: 9.0 +/- 3.8; I Bond: 3.0 +/- 1.5; Bond Force: 14 +/- 3.9; Futurabond: 8.8 +/- 3.8). Failure mode was predominantly adhesive. Conclusion: The bond strength of the self-etching systems tested was not significantly affected by the application of a hydrophobic layer, but a significant improvement was observed in AdheSE.

Alteration of Ca(2+)-ATPase activity in the homogenate, plasma membrane and microsomes of the salivary glands of streptozotocin-induced diabetic rats

Diabetes has been implicated in the dryness of the mouth, loss of taste sensation, sialosis, and other disorders of the oral cavity, by impairment of the salivary glands. The aim of the present study was to examine the plasma membrane, microsomal, and homogenate Ca(2+)-ATPase activity in the rat submandibular and parotid salivary glands of streptozotocin-induced diabetes. We have also examined the influence of the acidosis state oil this parameter. Diabetes was induced by an intraperitoneal injection of streptozotocin and acidosis was induced by daily injection of NH(4)Cl. At 15 and 30 days after diabetes induction, the animals were euthanized and the submandibular and parotid salivary glands were removed and analyzed. Ca(2+)-ATPase (total, independent, and dependent) was determined in the homo-enate, microsomal, and plasma membranes of the salivary glands of diabetic and control rats. Calcium concentration was also determined in the glands and showed to be hi-her in the diabetic animals. Ca(2+)-ATPase activity was found to be reduced in all cell fractions studied in the diabetic animals compared with control. Similar results were obtained for the submandibular salivary glands of acidotic animals; however in the parotid salivary glands it was found an increase in the enzyme activity. Copyright (c) 2009 John Wiley & Sons, Ltd.

Healing of critical-size cranial defects in guinea pigs using a bovine bone-derived resorbable membrane

Purpose: To investigate the healing of critical-size cranial bone defects (9-mm-diameter) in guinea pigs treated with a bovine bone-derived resorbable membrane. Materials and Methods: A sample of 42 guinea pigs was divided into test (n = 20), control (n = 20), and standard (n = 2) groups. A full-thickness trephine defect was made in the fronto-parietal bone of each animal. In the test group, the internal and external openings of the defect were each closed with a separate membrane, and the space between them was filled with blood clot and a central spacer. In the control group, the defect was filled only with the blood clot and spacer. At 1, 3, 6, and 9 months later, the calvarias (5 per period) for both the test and control groups were collected, fixed, radiographed, and histologically processed. The Standard-group animals were sacrificed immediately after surgery and used to determine the initial size of defect radiographically. The areas of defects in the radiographs were measured with image-analysis software and were compared between groups and periods by multiple regression analysis with the Bonferroni correction. Results: At 1 and 3 months, newly formed woven bone was histologically observed in both test and control groups. Radiographically, this new bone occupied an average of 32% of the defect area at 1 month and 60% at 3 months in the test group. In the control group, 21% of the defect was filled at 1 month and 39% at 3 months. However, the differences between treatments were not statistically significant (P > .05). At 6 and 9 months, a significant increase in newly formed lamellar bone was seen histologically in both groups. Radiographically, for the test group, the new bone occupied an average of 82% of the defect area at 6 months and 96% at 9 months. For the control group, new bone composed an average of 45% of the defect area at 6 months and 40% at 9 months. The differences between the test and control groups were statistically significant at 6 and 9 months (P < .05). Complete or almost complete filling of the defect was observed in several cases. Conclusion: It was concluded that the bovine bone-derived membrane is highly biocompatible and is able to promote good healing of critical-size defects in calvaria of guinea pig.

Biological monitoring of a xenomaterial for grafting: an evaluation in critical-size calvarial defects

Our purpose was to evaluate the osteoconduction potential of mixed bovine bone (MBB) xenografts as an alternative for bone grafting of critical-size defects in the calvaria of rats. After surgery, in the time intervals of 1, 3, 6, and 9 months, rats were killed and their skulls collected, radiographed and histologically prepared for analysis. The data obtained from histological analysis reported that the particles of MBB did not promote an intense immunological response, evidencing its biocompatibility in rats. Our results clearly showed the interesting evidence that MBB was not completely reabsorbed at 9 months while a small amount of newly formed bone was deposited by osteoprogenitor cells bordering the defect. However, this discrete bone-forming stimulation was unable to regenerate the bone defect. Overall, our results suggest that the properties of MBB are not suitable for stimulating intense bone regeneration in critical bone defects in rats.

Enamel Wetness Effects on Bond Strength Using Different Adhesive Systems

Objective: To evaluate, through the application of different dentin bonding systems, the influence of wetness on shear bond strength in enamel. Methods: This study evaluated three etch-and-rinse adhesive systems (Scotchbond MP [used with and without primer]; Singlebond; and Prime&Bond 2.1) and two self-etching adhesive systems (Clearfil SE Bond; and Xeno IV). Flat bovine enamel surfaces were either air-dried for 30 seconds or blotted with absorbent paper after acid-etching for the conventional bonding agents or before the application of self-etching bonding agents. The resin composite EsthetX was bonded to flat surfaces that had been treated with one of the adhesives, following the manufacturer`s instructions. After being stored in water at 37 degrees C for one week, bonded specimens were broken in shear. Data were evaluated with two-way analysis of variance (ANOVA) and Student-Newman-Keuls tests (alpha=0.05). For comparing each condition individually, regardless of the adhesive or wetness condition, a one-way ANOVA and a Student-Newman-Keuls test (alpha=0.05) were applied. Results: The two-way ANOVA showed significant differences among adhesive systems. An interaction effect was also observed (p < 0.05), but wetness did not influence shear bond strength (p=0.98). The one-way ANOVA showed that the all-in-one adhesive was the only material influenced by the presence of water at the enamel`s surface. Conclusion: The all-in-one adhesive behaved differently depending on whether the enamel surface was dry or wet.

SEM observation of the bond integrity of fiber-reinforced composite posts cemented into root canals

Objectives. To test the null hypothesis that continuity of resin cement/dentin interfaces is not affected by location along the root canal walls or water storage for 3 months when bonding fiber posts into root canals. Methods. Fiber posts were luted to bovine incisors using four resinous luting systems: Multilink, Variolink II, Enforce Dual and Enforce PV. After cementation, roots were longitudinally sectioned and epoxy resin replicas were prepared for SEM analysis (baseline). The original halves were immersed in solvent, replicated and evaluated. After 3 months water storage and a second solvent immersion, a new set of replicas were made and analyzed. The ratio (%) between the length (mm) of available bonding interface and the actual extension of bonded cement/dentin interface was calculated. Results. Significant lower percent values of bond integrity were found for Multilink (8.25%) and Variolink 11 (10.08%) when compared to Enforce Dual (25.11%) and Enforce PV (27.0%) at baseline analysis. The same trend was observed after immersion in solvent, with no significant changes. However, bond integrity was significantly reduced after 3 months water storage and a second solvent immersion to values below 5% (Multilink = 3.31%, Variolink=1.87%, Enforce Dual=1.20%, and Enforce PV=0.75%). The majority of gaps were depicted at the apical and middle thirds at baseline and after immersion in solvent. After 3 months, gaps were also detected at the cervical third. Significance. Bond integrity at the cement/dentin interface was surprisingly low after cementation of fiber posts to root canals with all resin cements. That was not significantly altered after immersion in solvent, but was further compromised after 3 months water storage. Gaps were mainly seen at middle and apical thirds throughout the experiment and extended to the cervical third after water storage for 3 months. Bond integrity of fiber posts luted to root canals was affected both by location and water storage. (C) 2007 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Cell compartmentalisation in planctomycetes: novel types of structural organisation for the bacterial cell

The organisation of cells of the planctomycete species Pirellula marina, Isosphaera pallida, Gemmata obscuriglobus, Planctomyces mat-is and Candidatus Brocadia anammoxidans was investigated based on ultrastructure derived from thin-sections of cryosubstituted cells, freeze-fracture replicas, and in the case of Gemmata obscuriglobus and Pirellllla marina, computer-aided 3-D reconstructions from serial sections of cryosubstituted cells. All planctomycete cells display a peripheral ribosome-free region, termed here the paryphoplasm, surrounding the perimeter of the cell, and an interior region including any nucleoid regions as well as ribosome-like particles, bounded by a single intracytoplasmic membrane (ICM), and termed the pirellulosome in Pirellula species. Immunogold labelling and RNase-gold cytochemistry indicates that in planctomycetes all the cell DNA is contained wholly within the interior region bounded by the ICM, and the paryphoplasm contains no DNA but at least some of the cell's RNA. The ICM in Isosphaera pallida and Planctomyces mat-is is invaginated such that the paryphoplasm forms a major portion of the cell interior in sections, but in other planctomycetes it remains as a peripheral zone. In the anaerobic ammonium-oxidising (anammox process) chemoautotroph Candidatus Brocadia anammoxidans the interior region bounded by ICM contains a further internal single-membrane-bounded region, the anam-moxosome. In Gemmata obscuriglobus. the interior ICM-bounded region contains the nuclear body, a double-membrane-bounded region containing the cell's nucleoid and all genomic DNA in addition to some RNA. Shared features of cell compartmentalisation in different planctomycetes are consistent with the monophyletic nature of the planctomycetes as a distinct division of the Bacteria. The shared organisational plan for the planctomycete cell constitutes a new type not known in cells of other bacteria.

A 24-month randomized clinical trial of a two- and three-step etch-and-rinse technique

Purpose This 24-month randomized paired tooth clinical study evaluated the performance of All Bond 3 used in the simplified (2-step) and full (3-step) versions Methods 33 patients, with at least two similar sized non-carious cervical lesions participated in this study A total of 66 restorations were placed, half using the 2-step All Bond 3 (AB3-2) and the other half using 3-step All Bond 3 (AB3-3) The restorations were placed incrementally using the composite resin Aelite The restorations were evaluated at baseline and after 6, 12 and 24 months following the modified USPHS criteria Statistical differences between the adhesive were tested using with McNemar`s test and clinical performance over time for each material with the Fisher`s exact test (alpha= 0 05) Results After 24 months, six AB3-2 and four AB3-3 were rated as bravo for marginal discoloration but did not differ from each other significantly (P> 0 05) The retention rates at 24 months of AB3-2 and AB3-3 were 90 9% and 97 0%, respectively (P> 0 05) (Am J Dent 2010,23 231-236)

Phylogeny of the filamentous bacterium 'Nostocoida limicola' III from activated sludge

Five strains of the filamentous bacterium 'Nostocoida limicola' III were successfully isolated into pure culture from samples of activated sludge biomass from five plants in Australia. 16S rRNA gene sequence analyses showed that all isolates were members of the Planctomycetales, most closely related to Isosphaera pallida, but they differed phenotypically from this species in that they did not glide and were not thermotolerant. The ultrastructure of these 'N. limicola' III isolates was also consistent with them being Planctomycetales, in that they possessed complex intracellular membrane systems compartmentalizing the cells. However, the arrangements of these intracellular membranes differed between isolates. These data confirm that 'N. limicola' III is phylogenetically unrelated to both 'N. limicola' I and 'N. limicola' II, activated sludge filamentous bacteria which share morphological features in common with 'N. limicola' III and which have been presumed historically to be the same or very similar bacteria.