CHARACTERIZATION OF Kiss1 NEURONS USING TRANSGENIC MOUSE MODELS

Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or beta-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Me lanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18-28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Lipins constitute a novel family of Mg2+-dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.

Vaccinating the vampire bat Desmodus rotundus against rabies

The objective of this study was to extend the previous work of indirect oral rabies immunization of vampire bats (Desmodus rotundus) maintained in captivity, which demonstrated the immunogenicity of the V-RG vaccine (Vaccinia-Rabies Glycoprotein) and indicated that although the results had been encouraging, a new method for concentrating the vaccine should be tested in order to avoid vaccine loss and increase the survival proportion of bats after rabies challenge. In this study, three groups of seven bats each were tested with vaccine concentrated by ultrafiltration through a cellulose membrane. The vaccine was homogenized in Vaseline paste and applied to the back of one vector bat, which was then reintroduced into its group. A dose of 10(5.0) MICLD50 rabies virus was used by intramuscular route to challenge the bats postvaccination. The survival proportion in the three groups after the challenge was 71.4%, 71.4% and 100%. (c) 2008 Elsevier B.V. All rights reserved.

Ivermectin impairs sexual behavior in sexually naive, but not sexually experienced male rats

Ivermectin (IVM) is an antiparasitic drug, widely used in domestic animals. In mammals, IVM act as a GABA agonist. This neurotransmitter has an important role in the regulation of sexual behavior. Thus, this study sought to investigate the effects of various medically relevant doses IVM on the sexual behavior of male rats. In particular, we also wished to examine if previous sexual experience modulated responses to IVM. In the first experiment, the sexual behavior of inexperienced male rats was analyzed after they received 0.2, 0.6, 1.0 or 2.0 mg/kg IVM, 15 mm prior to behavioral testing. In the second experiment, the effects of four previous sexual experiences on IVM treated rats (1.0 or 2.0 mg/kg, 15 min prior to the 5th session) were assessed. The standard therapeutic dose (0.2 mg/kg) did not impair the sexual behavior of inexperienced male rats. At a more concentrated dose (0.6 mg/kg), which is still within the therapeutic range, the appetitive phase of sexual behavior of inexperienced male rats was impaired. Likewise, 1.0 mg/kg impaired the appetitive phase. Previous sexual experience blocked almost entirely this sexual impairment, suggesting that previous sexual experience exerts a positive effect in attenuating the sexual impairment produced by IVM treatment. Therefore, the standard therapeutic dose of IVM can be used without producing side effects on sexual behavior. Use of more concentrated therapeutic doses is not recommended during reproductive periods, unless the animals have had previous sexual experience. (C) 2010 Elsevier Ltd. All rights reserved.

Oral lesions and immune reconstitution syndrome in HIV+/AIDS patients receiving highly active antiretroviral therapy. Epidemiological evidence

Objective: To determine whether opportunistic oral infections associated to HIV infection (OOI-HIV) are found in HIV+/AIDS patients with immune reconstitution related to highly active antiretroviral therapy (HAART). Methods. From among 1100 HIV+/AIDS patients (Service of Internal Medicine, Carlos Haya Hospital, Malaga, Spain) subjected to review of the oral cavity between January 1996 and May 2007, we identified those examined in 1996 and which were again examined between 1997 and 2007, and were moreover receiving HAART. The following data were collected: age, gender, form of contagion, antiretroviral therapy at the time of review, number of CD4+ lymphocytes/ml, and viral load (from 1997 onwards). We identified those subjects with an increase in CD4+ lymphocytes/ ml associated to HAART, and classified them as subjects with quantitative evidence of immune reconstitution (QEIR). Among these individuals with QEIR we moreover identified those with undetectable viral loads (QEIR+VL), and differentiated those patients with an increase in CD4+ lymphocytes > 500/ml (QEIRm+VL). In each group we determined the prevalence of OOI-HIV, following the diagnostic recommendations of the EC-Clearinghouse (CDC-Atlanta, USA - WHO). In addition, we analyzed the prevalence of OOI-HIV in the different groups in relation to the duration of HAART. Results. A total of 86 subjects were included (44 females and 42 males; 19 heterosexuals, 34 male homosexuals, and 33 intravenous drug abusers). Forty-two patients showed QEIR: 21 belonged to the QEIR+VL group, and 17 conformed the QEIRm+VL group. The prevalence of OOI-HIV per group was as follows: QEIR = 54.8%; QEIR+VL = 33%; QEIRm+VL = 35%. The most prevalent lesion in all groups was erythematous candidiasis. OOI-HIV increased with the duration of HAART (p=0.008), and were seen to be dependent upon late appearance of the mycotic lesions ( after 24 months under HAART). Conclusions: It is suggested that opportunistic oral infections associated to HIV infection form part of the clinical picture of immune reconstitution inflammatory syndrome, though such infections are of late onset.

Effect of ion exchange on R-curve behavior of a dental porcelain

The objective of this study was to evaluate the effect of the ion exchange treatment on the R-curve behavior of a leucite-reinforced dental porcelain, testing the hypothesis that the ion exchange is able to improve the R-curve behavior of the porcelain studied. Porcelain disks were sintered, finely polished, and submitted to an ion exchange treatment with a KNO(3) paste. The R-curve behavior was assessed by fracturing the specimens in a biaxial flexure design after making Vickers indentations in the center of the polished surface with loads of 1.8, 3.1, 4.9, 9.8, 31.4, and 49.0 N. The results showed that the ion exchange process resulted in significant improvements in terms of fracture toughness and flexural strength as compared to the untreated material. Nevertheless, the rising R-curve behavior previously observed in the control group disappeared after the ion exchange treatment, i.e., fracture toughness did not increase with the increase in crack size for the treated group.

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.

Mechanical Testing of Indirect Composite Materials Directly Applied on Implant Abutments

Purpose: To test the strength to failure and fracture mode of three indirect composite materials directly applied onto Ti-6Al-4V implant abutments vs cemented standard porcelain-fused-to-metal (PFM) crowns. Materials and Methods: Sixty-four locking taper abutments were randomly allocated to four groups and were cleaned in ethanol in an ultrasonic bath for 5 min. After drying under ambient conditions, the abutments were grit blasted and a custom 4-cusp molar crown mold was utilized to produce identical crowns (n = 16 per group) of Tescera (Bisco), Ceramage (Shofu), and Diamond Crown (DRM) according to the manufacturer`s instructions. The porcelain-fused-to-metal crowns were fabricated by conventional means involving the construction and a wax pattern and casting of a metallic coping followed by sintering of increasing layers of porcelain. All crowns were loaded to failure by an indenter placed at one of the cusp tips at a 1 mm/min rate. Subsequently, fracture analysis was performed by means of stereomicroscopy and scanning electron microscopy. One-way ANOVA at 95% level of significance was utilized for statistical analysis. Results: The single load to failure (+/- SD) results were: Tescera (1130 +/- 239 N), Ceramage (1099 +/- 257 N), Diamond Crown (1155 +/- 284 N), and PFM (1081 +/- 243 N). Stereomicroscopy analysis showed two distinct failure modes, where the loaded cusp failed either with or without abutment/metallic coping exposure. SEM analysis of the fractures showed multiple crack propagation towards the cervical region of the crown below a region of plastic deformation at the indenter contact region. Conclusion: The three indirect composites and PFM systems fractured at loads higher than those typically associated with normal occlusal function. Although each material had a different composition and handling technique, no significant differences were found concerning their single load to fracture resistance among composite systems and PFM.

Monolithic CAD/CAM Lithium Disilicate Versus Veneered Y-TZP Crowns: Comparison of Failure Modes and Reliability After Fatigue

Purpose: The aim of this research was to evaluate the fatigue behavior and reliability of monolithic computer-aided design/computer-assisted manufacture (CAD/CAM) lithium disilicate and hand-layer-veneered zirconia all-ceramic crowns. Materials and Methods: A CAD-based mandibular molar crown preparation, fabricated using rapid prototyping, served as the master die. Fully anatomically shaped monolithic lithium disilicate crowns (IPS e.max CAD, n = 19) and hand-layer-veneered zirconia-based crowns (IPS e.max ZirCAD/Ceram, n = 21) were designed and milled using a CAD/CAM system. Crowns were cemented on aged dentinlike composite dies with resin cement. Crowns were exposed to mouth-motion fatigue by sliding a WC-indenter (r = 3.18 mm) 0.7 mm lingually down the distobuccal cusp using three different step-stress profiles until failure occurred. Failure was designated as a large chip or fracture through the crown. If no failures occurred at high loads (> 900 N), the test method was changed to staircase r ratio fatigue. Stress level probability curves and reliability were calculated. Results: Hand-layer-veneered zirconia crowns revealed veneer chipping and had a reliability of < 0.01 (0.03 to 0.00, two-sided 90% confidence bounds) for a mission of 100,000 cycles and a 200-N load. None of the fully anatomically shaped CAD/CAM-fabricated monolithic lithium disilicate crowns failed during step-stress mouth-motion fatigue (180,000 cycles, 900 N). CAD/CAM lithium disilicate crowns also survived r ratio fatigue (1,000,000 cycles, 100 to 1,000 N). There appears to be a threshold for damage/bulk fracture for the lithium disilicate ceramic in the range of 1,100 to 1,200 N. Conclusion: Based on present fatigue findings, the application of CAD/CAM lithium disilicate ceramic in a monolithic/fully anatomical configuration resulted in fatigue-resistant crowns, whereas hand-layer-veneered zirconia crowns revealed a high susceptibility to mouth-motion cyclic loading with early veneer failures. Int J Prosthodont 2010; 23: 434-442.

Photoelastic Study of the Support Structures of Distal-Extension Removable Partial Dentures

Purpose: The double system of support, in which the distal-extension removable partial denture adapts, causes inadequate stress around abutment teeth, increasing the possibility of unequal bone resorption. Several ways to reduce or more adequately distribute the stress between abutment teeth and residual ridges have been reported; however, there are no definitive answers to the problem. The purpose of this study was to analyze, by means of photoelasticity, the most favorable stress distribution using three retainers: T bar, rest, proximal plate, I bar (RPI), and circumferential with mesialized rest. Materials and Methods: Three photoelastic models were made simulating a Kennedy Class II inferior arch. Fifteen dentures with long saddles, five of each design, were adjusted to the photoelastic patterns and submitted first to uniformly distributed load, and then to a load localized on the last artificial tooth. The saddles were then shortened and the tests repeated. The quantitative and qualitative analyses of stress intensity were done manually and by photography, respectively. For intragroup analyses the Wilcoxon test for paired samples was used, while for intergroup analyses Friedman and Wilcoxon tests were used to better identify the differences (p < 0.05). Results: The RPI retainer, followed by the T bar, demonstrated the best distribution of load between teeth and residual ridge. The circumferential retainer caused greater concentration of stress between dental apexes. Stress distribution was influenced by the type of retainer, the length of the saddle, and the manner of load application. Conclusions: The long saddles and the uniformly distributed loads demonstrated better distribution of stress on support structures.

Bleaching Agents with Varying Concentrations of Carbamide and/or Hydrogen Peroxides: Effect on Dental Microhardness and Roughness

To evaluate the effect of low and highly concentrated bleaching agents on microhardness and surface roughness of bovine enamel and root dentin. According to a randomized complete block design, 100 specimens of each substrate were assigned into five groups to be treated with bleaching agents containing carbamide peroxide (CP) at 10% (CP10); hydrogen peroxide (HP) at 7.5% (HP7.5) or 38% (HP38), or the combination of 18% of HP and 22% of CP (HP18/CP22), for 3 weeks. The control group was left untreated. Specimens were immersed in artificial saliva between bleaching treatments. Knoop surface microhardness (SMH) and average surface roughness (Ra) were measured at baseline and post-bleaching conditions. For enamel, there were differences between bleaching treatments for both SMH and Ra measurements (p = 0.4009 and p = 0.7650, respectively). SMH significantly increased (p < 0.0001), whereas Ra decreased (p = 0.0207) from baseline to post-bleaching condition. For root dentin, the group treated with CP10 exhibited the significantly highest SMH value differing from those groups bleached with HP18/CP22, HP7.5, which did not differ from each other. Application of HP38 resulted in intermediate SMH values. No significant differences were found for Ra (p = 0.5975). Comparing the baseline and post-bleaching conditions, a decrease was observed in SMH (p < 0.0001) and an increase in Ra (p = 0.0063). Bleaching agents with varying concentrations of CP and/or HP are capable of causing mineral loss in root dentin. Enamel does not perform in such bleaching agent-dependent fashion when one considers either hardness or surface roughness evaluations. Bleaching did not alter the enamel microhardness and surface roughness, but in root dentin, microhardness seems to be dependent on the bleaching agent used.

Effect of simulated masticatory loading on the retention of stud attachments for implant overdentures

P>This study assessed the effect of simulated mastication on the retention of two stud attachment systems for 2-implants overdentures. Sixteen specimens, each simulating an edentulous ridge with implants and an overdenture were divided into two groups, according to the attachment system: Group I (Nobel Biocare ball-socket attachments) and Group II (Locator attachments). Retention forces were measured before and after 400 000 simulated masticatory loads in a customised device. Data were compared by two-way anova followed by Bonferroni test (alpha = 0 center dot 05). Group I presented significantly lower retention forces (Newtons) than Group II at baseline (10 center dot 6 +/- 3 center dot 6 and 66 center dot 4 +/- 16 center dot 0, respectively). However, differences were not significant after 400 000 loads (7 center dot 9 +/- 4 center dot 3 and 21 center dot 6 +/- 17 center dot 0). The number of cycles did not influence the measurements in Group I, whereas a non-linear descending curve was found for Group II. It was concluded that simulated mastication resulted in minor changes for the ball attachment tested. Nevertheless, it reduced the retention of Locator attachments to 40% of the baseline values, what suggests that mastication is a major factor associated with maintenance needs for this system.

Compartmentalization of Ras proteins

The Ras GTPases operate as molecular switches that link extracellular stimuli with a diverse range of biological outcomes. Although many studies have concentrated on the protein-protein interactions within the complex signaling cascades regulated by Ras, it is becoming clear that the spatial orientation of different Ras isoforms within the plasma membrane is also critical for their function. H-Ras, N-Ras and K-Ras use different membrane anchors to attach to the plasma membrane. Recently it has been shown that these anchors also act as trafficking signals that direct palmitoylated H-Ras and N-Ras through the exocytic pathway to the cell surface but divert polybasic K-Ras around the Golgi to the plasma membrane via an as yet-unidentified-route. Once at the plasma membrane, H-Ras and :K-Ras operate in different microdomains. K-Ras is localized predominantly to the disordered plasma membrane, whereas H-Ras exists in a GTP-regulated equilibrium between disordered plasma membrane and cholesterol-rich lipid rafts. These observations provide a likely explanation for the increasing number of biological differences being identified between the otherwise highly homologous Ras isoforms and raise interesting questions about the role membrane microlocalization plays in determining the interactions of Ras with its effecters and exchange factors.

Design, modelling and analysis of a six component force balance for hypervelocity wind tunnel testing

A combination of modelling and analysis techniques was used to design a six component force balance. The balance was designed specifically for the measurement of impulsive aerodynamic forces and moments characteristic of hypervelocity shock tunnel testing using the stress wave force measurement technique. Aerodynamic modelling was used to estimate the magnitude and distribution of forces and finite element modelling to determine the mechanical response of proposed balance designs. Simulation of balance performance was based on aerodynamic loads and mechanical responses using convolution techniques. Deconvolution was then used to assess balance performance and to guide further design modifications leading to the final balance design. (C) 2001 Elsevier Science Ltd. All rights reserved.

Neurology of ciguatera

Ciguatera is a widespread ichthyosarcotoxaemia with dramatic and clinically important neurological features. This severe form of fish poisoning may present with either acute or chronic intoxication syndromes and constitutes a global health problem. Ciguatera poisoning is little known in temperate countries as a potentially global problem associated with human ingestion of large carnivorous fish that harbour the bioaccumulated ciguatoxins of the photosynthetic dinoflagellate Gambierdiscus toxicus. This neurotoxin is stored in the viscera of fish that have eaten the dinoflagellate and concentrated it upwards throughout the food chain towards progressively larger species, including humans. Ciguatoxin accumulates in all fish tissues, especially the liver and viscera, of at risk species. Both Pacific (P-CTX-1) and Caribbean (C-CTX-1) ciguatoxins are heat stable polyether toxins and pose a health risk at concentrations above 0.1 ppb. The presenting signs of ciguatera are primarily neurotoxic in more than 80% of cases. Such include the pathognomonic features of postingestion paraesthesiae, dysaesthesiae, and heightened nociperception. Other sensory abnormalities include the subjective features of metallic taste, pruritis, arthralgia, myalgia, and dental pain. Cerebellar dysfunction, sometimes diphasic, and weakness due to both neuropathy and polymyositis may be encountered. Autonomic dysfunction leads to hypotension, bradycardia, and hypersalivation in severe cases. Ciguatoxins are potent, lipophilic sodium channel activator toxins which bind to the voltage sensitive (site 5) sodium channel on the cell membranes of all excitable tissues. Treatment depends on early diagnosis and the early administration of intravenous mannitol. The early identification of the neurological features in sentinel patients has the potential to reduce the number of secondary cases in cluster outbreaks.