Effects that bovine sperm cryopreservation using two different extenders has on sperm membranes and chromatin

The process of cryopreservation impairs sperm cell function, potentially leading to a reduction in fertility. The objectives of the present study were to evaluate the effects that cryopreservation using two different extenders has on sperm motility and mitochondrial function, as well as on the integrity of plasma membranes, acrosomal membranes and chromatin, using practical and objective techniques. The focus of the present study was to identify correlations between alterations in sperm membranes and sperm motility in cryopreserved bovine spermatozoa. Seven ejaculates were collected from eight Simmental bulls (n = 56). After collection, semen volume and concentration were assessed for purposes of dilution. Sperm motility was evaluated subjectively and by computer-assisted semen analysis, morphological characteristics were evaluated by differential interference microscopy, the integrity of plasma and acrosomal membranes, as well as mitochondrial function, were determined using a combination of fluorescent probes containing fluorescein isothiocyanate-Pisum sativum agglutinin, propidium iodide or 5,5`,6,6`-tetrachloro-1,1`,3,3`-tetraethylbenzimidazolearbocyanine iodide. Chromatin integrity was evaluated using the acridine orange technique. The semen was subsequently divided into two aliquots and diluted with one of two extenders (Bioxcell(R) or Botu-Bov(R)), after which both were packaged in 0.5 mL straws and frozen using an automated system. Two straws of semen from each treatment were thawed, and the semen parameters were evaluated as described above. Cryopreservation of sperm reduced motility, damaging plasma and acrosomal membranes, as well as decreasing mitochondrial function. The Botu-Bov(R) extender was more effective in preserving sperm motility and membrane integrity than was the Bioxcell(R) extender. (C) 2007 Elsevier B.V. All rights reserved.

Sequence variation of the alpha-lactalbumin gene in Holstein and Nellore cows

The alpha-lactalbumin is a subunit of lactose-synthase, an enzyme responsible for lactose production, a disaccharide that influences milk production. Sequence variations of bovine alpha-lactalbumin have been associated with differences in milk yield. This study aimed to analyze allelic frequency differences at position-1689 (g. AG) and+15 (g. AG) of the alpha-lactalbumin gene in Holstein (Bos taurus) and Nellore (Bos indicus) cows. Blood samples were analyzed from 34 Holstein, 104 Nellore, and 99 Dairy Nellore cows using PCR-RFLP. The different RFLP patterns were sequenced and a novel sequence variation on nucleotide-46 was identified. An adenine at this position was designated as the A allele and a guanine was designated B allele. The frequencies of alleles A-1689, A-46, and A+15 differed between Holstein and both Nellore breeds. The results show that differences in alpha-lactalbumin allelic variants in the 5`-flanking and the 5`-UTR region might be associated with differences in milk production between Holstein cows and cows from Nellore breeds. However, the lack of difference between Nellore and Dairy Nellore suggests that other sequence variantions that regulate milk production might be responsible for the selection of Dairy Nellore cows with superior milk production.

Density of the Near Threatened jaguar Panthera onca in the caatinga of north-eastern Brazil

We report the first estimate of jaguar density in the semi-arid caatinga biome of north-eastern Brazil. During August-October 2007, in the Serra da Capivara National Park, we used camera traps to identify and count jaguars. Jaguar abundance and density were calculated using mark-recapture models. In a sampling effort of 1,249 camera-trap-nights we identified 12 adult jaguars and estimated an 2 abundance of 14 +/- 3.6 jaguars in an area of 524 km(2), i.e. a density of 2.67 +/- 1.00 jaguars per 100 km(2). This estimate is higher than in most other Brazilian biomes and indicates Serra da Capivara National Park as an important reserve for protecting jaguars in north-eastern Brazil.

Influence of oil contamination on in vitro bond strength of bonding agents to dental substrates

Purpose: To evaluate the influence of cleaning procedures (pumice, anionic detergent and both procedures together) on the tensile bond strength of etch-and-rinse and self-etch adhesive systems to bovine enamel and dentin in vitro. Methods: Eighty non-carious, bovine incisors were extracted, embedded in acrylic resin to obtain enamel/dentin specimens. Flat bonding surfaces were obtained by grinding. Groups were divided according to substrate (enamel or dentin), adhesive system [etch-and-rinse, Adper Single Bond 2 (SB) or self-etch, Clearfil Protect Bond (PB)]; and cleaning substances (pumice, anionic detergent and their combination). The teeth were randomly divided into 20 groups (n=8): G1 - Enamel (E) + SB; G2 -E + oil (O) + SB; G3 - E + O + Pumice (P) + SB; G4 - E + O + Tergentol (T) + SB; G5 - E + O + P + T + SB; G6 - E + PB; G7 - E + O + PB; G8 - E + O + P + PB; G9 - E + O + T + PB; GIO - E + O + P + T + PB; G11 - Dentin (D) + SB; G12 D + SB + O; G13 - D + SB + O + P; G14 - D + SB + O + T; G15 - D + SB + O + P + T; G16 - D + PB; G17 - D + O + PB +; G18 - D + O + P + PB; G19 - D + O + T + PB; G20 - D + O + P + T + PB. Specimens were contaminated with handpiece oil for 5 seconds before bonding. Adhesive systems and resin composite were applied according to manufacturers` instructions. Specimens were tested in tension after 24 hours of immersion using a universal testing machine at a crosshead speed of 0.5 mm/minute. Bond strengths were analyzed with ANOVA. Failure sites were observed and recorded. Results: Tensile bond strength in MPa were: G1 (23.6 +/- 0.9); G2 (17.3 +/- 2.2); G3 (20.9 +/- 0.9); G4 (20.6 +/- 0.5); G5 (18.7 +/- 2.3); G6 (23.0 +/- 1.0); G7 (21.5 +/- 2.4); G8 (19.9 +/- 1.3); G9 (22.1 +/- 1.2); G10 (19.1 +/- 1.2); G11 (18.8 +/- 1.3); G12 (15.7 +/- 2.1); G13 (17.8 +/- 3.3); G14 (15.3 +/- 2.9); G15 (15.6 +/- 1.9); G16 (14.7 +/- 2.3); G17 (5.5 +/- 0.9); G18 (19.3 +/- 1.8); G19 (15.6 +/- 1.6); G20 (20.3 +/- 3.9). Statistical analysis showed that the main factors substrate and cleaning were statistically significant, as well as the triple interaction between factors of variance. However, the factor adhesive system did not show statistical difference. Oil contamination reduced bond strengths, being less detrimental to enamel than to dentin. Etch-and-rinse (SB) and two-step self-etch (PB) systems had similar bond strengths in the presence of oil contamination. For etch-and-rinse (SB), the cleaning procedures were able to clean enamel, but dentin was better cleaned by pumice. When self-etch (PB) system was used on enamel, anionic detergent was the best cleaning substance, while on dentin the tested procedures were similarly efficient.

Mucocele in pediatric patients: Analysis of 36 children

Mucoceles are common benign lesions of the oral cavity that develop following extravasation or retention of mucous material from major or minor salivary glands. Most series report a higher incidence of mucocele in young patients but no studies exist for this specific age group. The records of 104 patients presenting with mucocele who underwent surgery in the Oral Diseases Clinic of the Department of Dermatology of the University of Sao Paulo, Brazil, between 1991 and 2006, were reviewed. Of these, 36 (34.6%) were 15 years old or younger, the youngest being 2 years old. Twenty-six patients were girls, 10 were boys. Lesions were located on the lower labial mucosa (30 patients), tongue (three), floor of the mouth (one), buccal mucosa (one), and in one patient the location was unknown. Histopathology revealed an extravasation pattern in 35 patients and a retention pattern in one. Of the distinct subtypes of mucocele, tongue lesions (mucocele of the glands of Blandin-Nuhn) seem to be more prevalent and retention mucoceles seem to be very rare in children.

A critical view on biaxial and short-beam uniaxial flexural strength tests applied to resin composites using Weibull, fractographic and finite element analyses

Objective. To evaluate the biaxial and short-beam uniaxial strength tests applied to resin composites based upon their Weibull parameters, fractographic features and stress distribution. Methods. Disk- (15 mm x 1 mm) and beam-shaped specimens (10 mm x 2 mm x 1 mm) of three commercial composites (Concept/Vigodent, CA; Heliomolar/Ivoclar-Vivadent, HE; Z250/3M ESPE, FZ) were prepared. After 48h dry storage at 37 degrees C, disks and beams were submitted to piston-on-three-balls (BI) and three-point bending (UNI) tests, respectively. Data were analyzed by Weibull statistics. Fractured surfaces were observed under stereomicroscope and scanning electron microscope. Maximum principal stress (sigma(1)) distribution was determined by finite element analysis (FEA). Maximum sigma(1-BI) and sigma(1-UNI) were compared to FZ strengths calculated by applying the average failure loads to the analytical equations (sigma(a-BI) and sigma(a-UNI)). Results. For BI, characteristic strengths were: 169.9a (FZ), 122.4b (CA) and 104.8c (HE), and for UNI were: 160.3a (FZ), 98.2b (CA) and 91.6b (HE). Weibull moduli ( m) were similar within the same test. CA and HE presented statistically higher m for BI. Surface pores ( BI) and edge flaws ( UNI) were the most frequent fracture origins. sigma(1-BI) was 14% lower than sigma(a-BI.) sigma(1-UNI) was 43% higher than sigma(a-UNI). Significance. Compared to the short-beam uniaxial test, the biaxial test detected more differences among composites and displayed less data scattering for two of the tested materials. Also, biaxial strength was closer to the material`s strength estimated by FEA. (C) 2009 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Influence of Photoactivation Protocol and Light Guide Distance on Conversion and Microleakage of Composite Restorations

Purpose: To evaluate the effect of light guide distance and the different photoactivation methods on the degree of conversion (DC) and microleakage of a composite. Methods and Materials: Three photoactivation protocols (600mW/cm(2) x 40 seconds; 400 mW/cm(2) x 60 seconds or 200 mW/cm(2) x 20 seconds, followed by 500 mW/cm(2) X 40 seconds) and three distances from the light source (0, 3 or 7 mm) were tested. Cylindrical specimens (5 nun diameter; 2 mm tall; n=3) were prepared for the DC test (FT-Raman). Class V cavities were made in 90 bovine incisors to conduct the microleakage test. The specimens were conditioned for 15 seconds with phosphoric acid (37%), followed by application of the adhesive system Prime & Bond NT (Dentsply/Caulk). The preparations were restored in bulk. The specimens were stored for 24 hours in distilled water (37 degrees C) before being submitted to the silvernitrate microleakage protocol. The restorations were sectioned and analyzed under 25x magnification. Results: Statistical analyses (two-way ANOVAs and Tukey test, alpha=0.05) found significance only for the factor distance (p=0.015) at the top of the composite for the DC test. Conversion was statistically lower for the 7 mm groups compared to the 0 and 3 mm groups, which were equivalent to each other. At the bottom of the specimens, none of the factors or interactions was significant (p<0.05). The Kruskal-Wallis test showed that, in general, the soft-start method led to lower microleakage scores when compared to the continuous modes, mainly when associated with a distancing of 7 mm (p<0.01). With the exception of specimens irradiated with 400mW/cm(2) that did not demonstrate variations on scores for the distances tested, higher microleakage was observed for shorter distances from the light source. Conclusions: Soft-start methods may reduce microleakage when the light guide distancing provides a low level of irradiance, which also causes a discrete reduction in the DC.

GP5+/6+ SYBR Green methodology for simultaneous screening and quantification of human papillomavirus

Background: Detection and quantification of human papillomavirus (HPV) may help in predicting the evolution of HPV infection and progression of associated lesions. Objectives: We propose a novel protocol using consensus primers GP5+/6+ in a SYBR Green quantitative real-time (Q-RT) polymerase chain reaction (PCR). The strategy permits screening for HPV infection and viral load quantification simultaneously. Study design: DNA from 153 archived cervical samples, previously tested for HPV detection by GP5+/6+ PCR and typed by EIA-RLB (enzyme immunoassay-reverse line blot) or sequence analysis, was analysed using SYBR Green Q-RT PCR. Melting temperature assay (T(m)) and cycle threshold (C(t)) were used to evaluate HPV positivity and viral load. The T(m) in the range of 77-82 degrees C was considered to be positive for HPV-DNA. HPV results generated through GP5+/6+ conventional PCR were considered the gold standard against which sensitivity and specificity of our assay were measured. Results: Out of 104 HPV positive samples, 100 (96.2%) were also determined as positive by SYBR Green Q-RT PCR; of the 49 HPV-negative samples, all were determined as negative. There was an excellent positivity agreement (K = 0.94) between the SYBR Green Q-RT and the previous methods employed. The specificity and sensitivity were 100% and 96.2%, respectively. Comparison of SYBR Green Q-RT and TaqMan oligo-probe technologies gave an excellent concordance (pc = 0.95) which validated the proposed strategy. Conclusions: We propose a sensitive and easy-to-perform technique for HPV screening and viral load quantification simultaneously. (C) 2009 Elsevier B.V. All rights reserved.

Progressive recovery of osseoperception as a function of the combination of implant-supported prostheses

The extraction of teeth involves the elimination of extremely sensitive periodontal mechanoreceptors, which play an important role in oral sensory perception. Objectives: The aim of this study was to evaluate the recovery of interocclusal sensory perception for micro-thickness in individuals with different types of implant-supported prostheses. Materials and Methods: Wearers of complete dentures (CDs) comprised the negative control group (group A, n=17). The experimental group consisted of wearers of prostheses supported by osseointegrated implants (Group B, n=29), which was subsequently divided into 4 subgroups: B(1) (n=5) - implant supported overdentures (ISO) occluding with CD; B(2) (n=6) - implant-supported fixed prostheses (ISFP) occluding with CD; B3 (n=8) - wearers of maxillary and mandibular ISFP, and B(4) (n=10) - ISFP occluding with natural dentition (ND). Individuals with ND represented the positive control group (Group C, n=24). Aluminum foils measuring 10 mu m, 24 mu m, 30 mu m, 50 mu m, 80 mu m, and 104 mu m thickness were placed within the premolar area, adding up to 120 tests for each individual. Results: The mean tactile thresholds of groups A, B(1), B(2), B(3), B(4), and C were 92 mu m, 27 mu m, 27 mu m, 14 mu m, 10 mu m, and 10 mu m, respectively. [Correction added after publication online 18 April 2008: in the preceding sentence 92 mu m, 27 mu m, 14 mu m, 10 mu m and 10 mu m, was corrected to 92 mu m, 27 mu m, 27 mu m, 14 mu m, 10 mu m and 10 mu m.] The Kruskal-Wallis test revealed significant difference among groups (P < 0.05). The Dunn test revealed that group A was statistically different from groups C, B(3), and B(4), and that B(1) and B(2) were statistically different from group C. Conclusion: Progressive recovery of osseoperception as a function of the combination of implant-supported prostheses could be observed. Moreover, ISO and/or ISFP combinations may similarly maximize the recovery of osseoperception.

High frequency of low-level microsatellite instability in early colorectal cancer

Molecular events in early colorectal cancers (CRCs) have not been well elucidated because of the low incidence of early CRCs in clinical practice. Therefore, we studied 104 sporadic early CRCs with invasion limited to submucosa compared with 116 advanced CRCs. Loss of heterozygosity as well as microsatellite instability (MSI) status was examined. A significantly high frequency of low-level MSI (MSI-L) phenotype was detected in early CRCs (51.0%) compared with advanced CRCs (25.9%; P = 0.0001). In early and advanced CRCs, samples with MSI-L phenotype differed from microsatellite stable (MSS) phenotype with respect to loss of heterozygosity at 1p32 and 8p12-22. MSI-L is a frequent genetic event in early CRCs and may be a novel pathway in colorectal carcinogenesis distinct from both MSI-H and MSS.

Ionotropic glutamatergic receptors in the rostral medullary raphe modulate hypoxia and hypercapnia-induced hyperpnea

It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia and hypercapnia. We assessed the role of ionotropic glutamate receptors in the rostral MR (rMR) in the respiratory responses to hypoxia and hypercapnia by measuring pulmonary ventilation (V(E)) and body temperature (Tb) of male Wistar rats before and after microinjecting Kynurenic acid (KY, an ionotropic glutamate receptors antagonist, 0.1 mM) into the rMR followed by 60 min of hypoxia (7% O(2)) or hypercapnia exposure (7% CO(2)). Compared to the control group, the ventilatory response to hypoxia was attenuated in animals treated with KY intra-rMR, however the ventilatory response to hypercapnia increased significantly. No differences in Tb among groups were observed during hypoxia or hypercapnia. These data suggest that the glutamate acting on ionotropic receptors in the rMR exerts an excitatory modulation on hyperventilation induced by hypoxia but an inhibitory modulation on the hypercapnia-induced hyperpnea. (C) 2010 Elsevier B.V. All rights reserved.

Effect of growth hormone on in vitro osteogenesis and gene expression of human osteoblastic cells is donor-age-dependent

it has been demonstrated that the effect of GH on bone tissue is reduced with aging. In this study we tested the hypothesis that the action of GH on osteoblastic cells is donor-age-dependent by investigating the effect of GH on the development of osteoblastic phenotype in cultures of cells from adolescents (13-16 years old), young adults (18-35 years old), and adults (36-49 years old). Osteoblastic cells derived from human alveolar bone were cultured with or without GH for periods of up to 21 days, and parameters of in vitro osteogenesis and gene expression of osteoblastic markers were evaluated. GH increased culture growth, collagen content and alkaline phosphatase (ALP) activity in cultures from adolescents and young adults, whereas non-significant effect was observed in cultures from adults. While GH significantly increased the bone-like formation in cultures from adolescents, a slightly effect was observed in cultures from young adults and no alteration was detected in cultures from adults. Results from real-time PCR demonstrated that GH upregulated ALP, osteocalcin, type I collagen, and Cbfa1 mRNA levels in cultures from adolescents. In addition, cultures from young adults showed higher ALP mRNA expression and the expression of all evaluated genes was not affected by GH in cultures from adults. These results indicate that the GH effect on both in vitro osteogenesis and gene expression of osteoblastic markers is donor-age-dependent, being more pronounced on cultures from adolescents.

Earth pressure on cantilever walls at design retained heights

There are many methods for the analysis and design of embedded cantilever retaining walls. They involve various different simplifications of the pressure distribution to allow calculation of the limiting equilibrium retained height and the bending moment when the retained height is less than the limiting equilibrium value, i.e. the serviceability case. Recently, a new method for determining the serviceability earth pressure and bending moment has been proposed. This method makes an assumption defining the point of zero net pressure. This assumption implies that the passive pressure is not fully mobilised immediately below the excavation level. The finite element analyses presented in this paper examine the net pressure distribution on walls in which the retained height is less, than the limiting equilibrium value. The study shows that for all practical walls, the earth pressure distributions on the front and back of the wall are at their limit values, Kp and K-a respectively, when the lumped factor of safety F-r is less than or equal to2.0. A rectilinear net pressure distribution is proposed that is intuitively logical. It produces good predictions of the complete bending moment diagram for walls in the service configuration and the proposed method gives results that have excellent agreement with centrifuge model tests. The study shows that the method for determining the serviceability bending moment suggested by Padfield and Mair(1) in the CIRIA Report 104 gives excellent predictions of the maximum bending moment in practical cantilever walls. It provides the missing data that have been needed to verify and justify the CIRIA 104 method.

Crystallization and preliminary X-ray diffraction studies of FHA domains of Dun1 and Rad53 protein kinases

Forkhead-associated (FHA) domains are modular protein–protein interaction domains of ~130 amino acids present in numerous signalling proteins. FHA-domain-dependent protein interactions are regulated by phosphorylation of target proteins and FHA domains may be multifunctional phosphopeptide-recognition modules. FHA domains of the budding yeast cell-cycle checkpoint protein kinases Dun1p and Rad53p have been crystallized. Crystals of the Dun1-FHA domain exhibit the symmetry of the space group P6122 or P6522, with unit-cell parameters a = b = 127.3, c = 386.3 Å; diffraction data have been collected to 3.1 Å resolution on a synchrotron source. Crystals of the N-terminal FHA domain (FHA1) of Rad53p diffract to 4.0 Å resolution on a laboratory X-ray source and have Laue-group symmetry 4/mmm, with unit-cell parameters a = b = 61.7, c = 104.3 Å.

Polymorphisms at the glutathione S-transferase GSTM1, GSTT1 and GSTP1 loci: risk of ovarian cancer by histological subtype

The phase II glutathione S-transferases (GSTs) GSTT1, GSTM1 and GSTP1 catalyse glutathione-mediated reduction of exogenous and endogenous electrophiles. These GSTs have broad and overlapping substrate specificities and it has been hypothesized that allelic variants associated with less effective detoxification of potential carcinogens may confer an increased susceptibility to cancer. To assess the role of GST gene variants in ovarian cancer development, we screened 285 epithelial ovarian cancer cases and 299 unaffected controls for the GSTT1 deletion (null) variant, the GSTM1 deletion (null) variant and the GSTP1 codon 104 A-->G Ile-->Val amino acid substitution variant, The frequencies of the GSTT1, GSTM1 and GSTP1 polymorphic variants did not vary with tumour behaviour (low malignant potential or invasive) or p53 immunohistochemical status. There was a suggestion that ovarian cancers of the endometrioid or clear cell histological subtype had a higher frequency of the GSTT1 and GSTM1 deletion genotype than other histological subgroups. The GSTT1, GSTM1 and GSTP1 genotype distributions did not differ significantly between unaffected controls and ovarian cancer cases (overall or invasive cancers only). However, the GSTM1 null genotype was associated with increased risk of endometrioid/clear cell invasive cancer [age-adjusted OR (95% CI) = 2.04 (1.01-4.09), P = 0.05], suggesting that deletion of GSTM1 may increase the risk of ovarian cancer of these histological subtypes specifically. This marginally significant finding will require verification by independent studies.