Isolation of a novel intertypic recombinant human mastadenovirus B2 from two unrelated bone marrow transplant recipients.

Human adenoviruses (HAdV) are well-known opportunistic pathogens of immunocompromised adult and pediatric patients but specific associations between HAdV species or individual HAdV types and disease are poorly understood. In this study we report the isolation of a novel HAdV-B2 genotype from two unrelated immunocompromised patients, both recipients of a hematopoietic cell transplant. In both patients, the course of HAdV infection is consistent with a scenario of reactivation of a latent virus rather than a primary opportunistic infection. Archived HAdV PCR-positive plasma, urine, and stool specimens were processed for virus isolation and detailed molecular characterization. Virus isolates were recovered from patient 1 from PCR-positive urine specimens obtained at days 103 and 116 after transplant in association with gross hematuria, and from a stool specimen obtained 138 days after transplant in association with diarrhea. An isolate was recovered from patient 2 from a PCR-positive urine specimen. Hexon and fiber gene amplification and sequencing were carried out for initial molecular typing, identifying the isolates as an intertypic recombinant with a HAdV-11-like hexon gene and a HAdV-77-like fiber gene. Comprehensive restriction fragment length polymorphism (RFLP) analysis was performed on viral DNA purified from urine and stool isolates, and next generation whole genome sequencing was carried out on purified viral genomic DNA. The genomes of the two isolated strains are 99.5% identical and represent the same RFLP genomic variant. The identified virus is a novel HAdV-B2 genotype designated HAdV-78 exhibiting a HAdV-11-like penton base, a HAdV-11-like hexon and a HAdV-77-like fiber (P11H11F77).

High-order dispersion mapping of an optical fiber.

We report on measurements of high-order dispersion maps of an optical fiber, showing how the ratio between the third and fourth-order dispersion (ß3/ß4) and the zero-dispersion wavelength (λ0) vary along the length of the fiber. Our method is based on Four-Wave Mixing between short pulses derived from an incoherent pump and a weak laser. We find that the variations in the ratio ß3/ß4 are correlated to those in λ0. We present also numerical calculations to illustrate the limits on the spatial resolution of the method. Due to the good accuracy in measuring λ0 and ß3/ß4 (10 -3% and 5% relative error, respectively), and its simplicity, the method can be used to identify fiber segments of good uniformity, suitable to build nonlinear optical devices such as parametric amplifiers and frequency comb generators.

Effect of Methacryloyloxydecyl Dihydrogen Phosphate-Containing Silane and Adhesive Used Alone or in Combination on the Bond Strength and Chemical Interaction With Zirconia Ceramics Under Thermal Aging.

CLINICAL RELEVANCE: Achieving durable bonding to zirconia is fundamental for the application of a methacryloyloxydecyl dihydrogen phosphate (MDP)-containing silane solution or an MDP-containing silane solution associated with an MDP-containing universal adhesive. SUMMARY: Objectives: To evaluate the effect of a methacryloyloxydecyl dihydrogen phosphate (MDP)-containing silane coupling agent and universal adhesive, used alone or in combination, on the microshear bond strength (µSBS) to zirconia after 24 hours of water storage (24h) and after 10,000 thermocycles (TC), complemented with chemical analysis of the surface to establish the presence of MDP on the surface of the zirconia after bonding procedures.Methods and Materials: Thirty computer-aided design/computed-aided manufacturing blocks of zirconia were cut into four sections (6×6×6 mm) and sintered. Zirconia sections (n=96) were assigned to 24 groups according to three factors: 1) silane (no silane, Monobond S [MBS], Monobond P [MB+]), 2) adhesive + resin cement (no adhesive + Enforce [ENF], no adhesive + RelyX Ultimate [REX], Prime&Bond Elect + Enforce [PBE/ENF], Scotchbond Universal + RelyX Ultimate [SBU/REX]), and 3) thermocycling (no thermocycling [24h], 10,000 thermocycles [TC]). Upon silane/adhesive application, cylinder-shaped matrices were filled with resin cement and light cured. Specimens were tested in µSBS (1.0 mm/min) after 24h or TC. The µSBS data were analyzed using twoway ANOVA and Tukey's post hoc test (α=0.05). In addition, micro-Raman spectroscopy was used to analyze the zirconia surface for immediate chemical interaction analysis (n=24).Results: For the 24h condition, PBE/ENF resulted in lower mean µSBS than both groups with silane without PBE (MBS and MB+ groups; p<0.001). SBU alone or MB+ alone and MB+ associated with SBU showed the highest mean µSBS (p<0.001). For the TC condition, all groups showed a significant decrease in mean µSBS compared with those of 24h (p<0.001), with the exception of MB+ associated to SBU (p>0.05). However, the application of MB+ alone or MB+ associated to SBU resulted in higher mean µSBS (p<0.001) after TC than the remaining TC groups. In terms of chemical interaction, only the SBU groups, alone or combined with both of the silane agents, were associated with the methacrylate groups after rinsing.Conclusions: The results of the current study support the use of an MDP-containing silane solution or an MDP-containing silane solution associated with an MDP-containing universal adhesive for bonding to air-abraded zirconia, as a more stable bonding after thermocycling.

Expression of Toll-like receptors 2 and 4 and its association with matrix metalloproteinases in symptomatic and asymptomatic apical periodontitis.

To determine Toll-like receptors (TLR)2 and TLR4 expression levels and associate them with matrix metalloproteinases (MMPs) in asymptomatic apical periodontitis (AAP), symptomatic apical periodontitis (SAP), and healthy controls. Apical tissue/lesion samples were obtained from chronic AAP (n = 35) and SAP (n = 29), and healthy periodontal ligament (HPL, n = 10) with indication of tooth extraction, respectively. mRNA expression levels of TLR2, TLR4, MMP-1, MMP-2, MMP-8, and MMP-13 were determined by real-time reverse-transcription polymerase chain reaction. The data were analyzed with Kruskal-Wallis and Dunn's pot hoc test (p < 0.05). The correlation coefficient was obtained using the Spearman correlation (p < 0.05). TLR2, MMP-1, MMP-2, and MMP-13 mRNA levels were the highest in SAP followed by AAP and controls (p < 0.05). TLR4 and MMP-8 were over expressed in AAP and SAP compared to HPL (p < 0.05). TLR2 positively correlated with TLR4, MMP-1, MMP-8, and MMP-13 in SAP (p < 0.05). TLR2 and TLR4 are overexpressed in apical lesions versus healthy periodontal ligament and correlate with collagenolytic MMPs. Particularly, TLR2 is overexpressed in SAP in association with MMP-1, MMP-8, and MMP-13. Our results suggest that the activation of TLR2 along with MMP overexpression might contribute to SAP clinical presentation and progression. TLRs, MMPs, and their interaction can explain the clinical presentations and evolution of apical periodontitis and might represent key targets for new diagnostic and treatment approaches.

Suitability of multiple Mediterranean oak species as a food resource for Reticulitermes grassei Clément (Isoptera: Rhinotermitidae).

The subterranean termite Reticulitermes grassei Clément causes lesions in the trunk of Quercus suber L. by constructing feeding galleries, but no information is available regarding other Quercus species from the Mediterranean region. This work aimed to study the suitability of the other main oak species of Mediterranean forests as a food resource for R. grassei. Two experiments, choice and non-choice feeding, were conducted lasting for 15, 30, and 45 days each. In the non-choice experiment, termites were offered one of the following food types: Quercus suber, Quercus ilex L., Quercus faginea Lam, cork or Pinus pinea L., which was considered the control. The choice feeding experiment used all the same food types listed above, supplied simultaneously in the same container. Food selection was examined by analysing the relationships over time between surviving termites and food consumption. The results indicated that R. grassei could be considered a generalist species, as it consumed the cork and wood of all oak species, as well as displaying a clear preference for soft wood (pine). Correlation analysis indicated that consumption was not dependent on wood density. Survival of R. grassei was influenced by the time of exposure to different oak species, but a high survival rate was maintained over time in the pine treatment (upper 70% in the three experiments). Given these results, it can be concluded that all the oak species are a suitable food source for R. grassei.

Autecology, feeding preferences and reproductive biology of Chorthippus (Glyptobothrus) vagans (Eversmann, 1848) (Orthoptera: Gomphocerinae) in Mediterranean ecosystems.

Chorthippus vagans is a common species of Gomphocerinae (Orthoptera) on the Iberian Peninsula. It is endangered in Central Europe where information about its ecological requirements is available; however, aspects of its biology are almost unknown in Mediterranean ecosystems, where it is widespread and common. The focus of this study was to determine how C. vagans adjusts its biology to environmental features of this ecosystem and to interpret how it may be affected by the ecological changes related to the re-vegetation programme linked to the construction of the Breña dam (SW Spain). The research parameters included the autoecology, feeding response and some aspects of reproduction of this species in the Southern Iberian Peninsula. To determine the local distribution and phenology of C. vagans, monthly samplings were conducted (2007-2010) in 12 sampling plots. For the food selection tests, ten nymphs and ten adults were placed individually in insectaries under controlled conditions. Grasses (Lolium sp.) and shrubs (Cistus sp.) were supplied ad libitum in two types of tests, monospecific and mixed diet. The reproductive biology was analysed by both observations of anatomical structures (integument, bristles, tibial spines, tarsal claws and mandibles) and ovarian dissections of 29 females and in laboratory rearing experiments with 15 pairs of adults. The results indicate that C. vagans shows an extended activity period which peaks at the end of summer. It is a polyphagous grasshopper, although adults show a slight preference for grasses. In addition, it is a univoltine species with spring-summer breeding activity.

Efficient graphene saturable absorbers on D-shaped optical fiber for ultrashort pulse generation.

We demonstrated a method to construct high efficiency saturable absorbers based on the evanescent light field interaction of CVD monolayer graphene deposited on side-polished D-shaped optical fiber. A set of samples was fabricated with two different core-graphene distances (0 and 1 µm), covered with graphene ranging between 10 and 25 mm length. The mode-locking was achieved and the best pulse duration was 256 fs, the shortest pulse reported in the literature with CVD monolayer graphene in EDFL. As result, we find a criterion between the polarization relative extinction ratio in the samples and the pulse duration, which relates the better mode-locking performance with the higher polarization extinction ratio of the samples. This criterion also provides a better understanding of the graphene distributed saturable absorbers and their reproducible performance as optoelectronic devices for optical applications.

Influence of Conditioning Time of Universal Adhesives on Adhesive Properties and Enamel-Etching Pattern.

OBJECTIVES: To evaluate the effect of application protocol in resin-enamel microshear bond strength (µSBS), in situ degree of conversion, and etching pattern of three universal adhesive systems. METHODS AND MATERIALS: Sixty-three extracted third molars were sectioned in four parts (buccal, lingual, and proximals) and divided into nine groups, according to the combination of the main factors-Adhesive (Clearfil Universal, Kuraray Noritake Dental Inc, Tokyo, Japan; Futurabond U, VOCO, Cuxhaven, Germany; and Scotchbond Universal Adhesive, 3M ESPE, St Paul, MN, USA)-and enamel treatment/application time (etch-and-rinse mode [ER], self-etch [SE] application for 20 seconds [SE20], and SE application for 40 seconds [SE40]). Specimens were stored in water (37°C/24 h) and tested at 1.0 mm/min (µSBS). The degree of conversion of the adhesives at the resin-enamel interfaces was evaluated using micro-Raman spectroscopy. The enamel-etching pattern was evaluated under a scanning electron microscope. Data were analyzed with two-way analysis of variance and Tukey test (α=0.05). RESULTS: In general, the application of the universal adhesives in the SE40 produced µSBS and degree of conversion that were higher than in the SE20 (p<0.01) and similar to the ER mode. The deepest enamel-etching pattern was obtained in the ER mode, followed by the SE40. CONCLUSIONS: The active and prolonged application of universal adhesives in the SE mode may be a viable alternative to increase the degree of conversion, etching pattern, and resin-enamel bond strength.

Impaired cholinergic function in cell lines derived from the cerebral cortex of normal and trisomy 16 mice.

Murine trisomy 16 is an animal model of human Down's syndrome. We have successfully established permanently growing cell lines from the cerebral cortex of normal and trisomy 16 foetal mice using an original procedure. These lines, named CNh (derived from a normal animal) and CTb (derived from a trisomic foetus), express neuronal markers. Considering that Down's syndrome exhibits cholinergic deficits, we examined cholinergic function in these lines, using incorporation of [3H]-choline and fractional release studies. After 1, 3 and 5 min of [3H]-choline incubation, CTb cell uptake was lower by approximately 50% compared to controls. Hemicholinium-3 significantly reduced the incorporation of [3H]-choline in both CNh and CTb cells at high concentration (10 microM), suggesting high-affinity choline transport. However, CTb cells exhibited greater sensitivity to the blocker. For fractional release experiments, the cells were stimulated by K+ depolarization, glutamate or nicotine. When depolarized, CTb cells showed a 68% reduction in fractional release of [3H]-acetylcholine compared to CNh cell line, and a 45% reduction when stimulated by nicotine. Interestingly, glutamate induced similar levels of release in both cell types. The results indicate the existence of cholinergic dysfunction in CTb cells when compared to CNh, similar to that reported for primary cultures of trisomy 16 brain tissue (Fiedler et al. 1994, Brain Res., 658, 27-32). Thus, the CTb cell line may serve as a model for the study of Down's syndrome pathophysiology.

Neurite outgrowth in developing mouse spinal cord neurons is modulated by glycine receptors.

The effect of glycine receptor activation on neurite outgrowth and survival was studied in 5 DIV (days in vitro) spinal neurons. These neurons were depolarized by spontaneous synaptic activity and by glycine, but not by glutamate. These responses were accompanied by increases in intracellular calcium concentration measured with Indo-1 and Fluo-3. Glycine (100 microM, 48 h) increased (46 +/- 6%) the number of primary neurites and total neuritic length. This effect was mediated by synaptic activity and calcium influx because TTX (1 microM) and nimodipine (4 microM) blocked the stimulatory effect of glycine. Neuronal survival, on the other hand, was not affected. This study shows for the first time the modulatory effect of glycine receptors on spinal neuron development.

Multiple calcium pathways induce the expression of SNAP-25 protein in chromaffin cells.

Incubation of bovine adrenal chromaffin cells in high K+ (38 mM) during 24-48 h enhanced 2.5 to five times the expression of SNAP-25 protein and mRNA, respectively. This increase was reduced 86% by furnidipine (an L-type Ca2+ channel blocker) but was unaffected by either omega-conotoxin GVIA (an N-type Ca2+ channel blocker) or -agatoxin IVA (a P/Q-type Ca2+ channel blocker). Combined blockade of N and P/Q channels with omega-conotoxin MVIIC did, however, block by 76% the protein expression. The inhibitory effects of fumidipine were partially reversed when the external Ca2+ concentration was raised from 1.6 to 5 mM. These findings, together with the fact that nicotinic receptor activation or Ca2+ release from internal stores also enhanced SNAP-25 protein expression, suggest that an increment of cytosolic Ca2+ concentration ([Ca2+]), rather than its source or Ca2+ entry pathway, is the critical signal to induce the protein expression. The greater coupling between L-type Ca2+ channels and protein expression might be due to two facts: (a) L channels contributed 50% to the global [Ca2+]i rise induced by 38 mM K+ in indo-1-loaded chromaffin cells and (b) L channels undergo less inactivation than N or P/Q channels on sustained stimulation of these cells.

Calcium signals in cell lines derived from the cerebral cortex of normal and trisomy 16 mice.

We established two immortalized cell lines from cerebral cortex of normal (CNh) and trisomy 16 (CTb) mouse fetuses, an animal model of human trisomy 21. Those cells loaded with the fluorescent Ca2+ dyes, Indo-1 and Fluo-3, exhibited increments of intracellular Ca2+ ([Ca2+]i) in response to external glutamate, NMDA, AMPA and kainate. CTb cells exhibited higher basal Ca2+ concentrations and had higher amplitude and slower time-dependent kinetics in the decay than CNh cells, suggesting an impaired Ca2+ buffering capacity in the trisomy 16-derived cell line. Nicotine also induced increments of [Ca2+]i. The CTb cell line could represent a model for studying cellular alterations related to Down syndrome.

Intracellular calcium and arachidonic acid increase SNAP-25 expression in cultured rat hippocampal explants, but not in cultured rat cerebellar explants.

The effects of the increase of intracellular calcium, induced by membrane depolarization with 50 mM KCl, and arachidonic acid (AA) on the expression of 25-kD synaptosomal-associated protein (SNAP-25) were studied in cultured rat hippocampal and cerebellar explants, and PC12 rat pheochromocytoma cells, using immunoblot analysis. Incubation periods of 24 h and 48 h in 50 mM KCl increased SNAP-25 levels in hippocampal explants and PC12 cells, but not on cerebellar explants. Otherwise, a 24 h incubation with 10 microM AA increased SNAP-25 expression only in hippocampal explants, although 100 ng/ml phorbol 12-myristate 13-acetate (PMA) did not have effect. These results indicate that intracellular calcium and AA can modulate the expression of SNAP-25, depending on the origin of the tissue.

Clinical evaluation of the effect of calcium-channel blockers on verbal learning.

The effect of verapamil and nimodipine on verbal learning was evaluated in a double-blind clinical trial. Thirty-seven healthy volunteers were distributed in three groups to receive a treatment with nimodipine, verapamil or placebo. Neither verapamil nor nimodipine modifies verbal learning as measured by the selective remembering test of Buschke and Fuld.

Effect of protein synthesis inhibitors on synexin levels and secretory response in bovine adrenal medullary chromaffin cells.

The effects of the protein synthesis inhibitors actinomycin D and cycloheximide on the cellular content of the calcium binding protein synexin, and on the secretory response of cultured bovine adrenal medullary chromaffin cells were determined. Both protein synthesis inhibitors produced a slow decrease in the cellular synexin content. The synexin level was reduced by 50% after 133 h of incubation in the presence of 2 micrograms/ml actinomycin D or 5 micrograms/ml cycloheximide. However, this was partly due to an artefactual stabilization of synexin, since metabolic labelling of synexin with [35S]methionine showed that the half-time of degradation was only 40 h. The secretory response of chromaffin cells was quickly diminished in the presence of protein synthesis inhibitors. Catecholamine secretion induced by membrane depolarization or barium stimulation of intact cells, or by calcium stimulation of digitonin-permeabilized cells was decreased by 77-82% after 24 h of incubation in the presence of 5 micrograms/ml cycloheximide. These results suggest that, in addition to synexin, at least one or more proteins with a shorter half-time of degradation than synexin are involved in the secretory response of adrenal chromaffin cells.

New 4-alkyl-1,4-dihydropyridines: evaluation of photostability and phototoxic potential.

The photostability and phototoxic potential of two new 4-alkyl-1,4-dihydropyridines (PCA-4230 and PCA-4248) were investigated. When these 4-alkyl-1,4-dihydropyridines were irradiated with a multilamp photoreactor (band centred at 350 nm), both exhibited a slow photodegradation showing first-order kinetics. The photodegradation rate constants were 0.37 h-1 for PCA-4248 and 0.39 h-1 for PCA-4230 in oxygenated conditions. The photodecomposition was slower for both drugs in the absence of oxygen. In order to evaluate the phototoxicity induced by these drugs, red blood cells and Hep-2 (human laringo carcinoma cell line) were irradiated using a minisolarium, which emits UVA radiation (350-390 nm). The results showed that PCA-4248 and PCA-4230 did not exhibit a phototoxic effect in the two models tested.

Lipid peroxidation and loss of potassium from red blood cells produced by phototoxic quinolones.

Alterations of the cationic permeability of red blood cell membranes induced by the photosensitiser nalidixic acid were demonstrated by evaluating the potassium loss from intact erythrocytes. The results show that an increase in intracellular potassium efflux, precedes the photohemolysis induced by nalidixic acid. The addition of a nonpermeable osmotic solute, such as sucrose, inhibited photohemolysis but not the potassium loss, indicating a colloid osmotic lysis. Lipid peroxidation induced by nalidixic acid and other photosensitiser quinolones (oxolinic acid and rosoxacin) was time irradiation-dependent. Although rosoxacin was the most photoperoxidative, none of the three quinolones studied produced significant lipid peroxidation. However, of the three quinolones studied, only rosoxacin considerably diminished the percentage of the cholesterol extracted from red blood cell membranes. It is postulated that the increased cation permeability induced by nalidixic and oxolinic acids cannot be attributed to cholesterol oxidation nor to lipid peroxidation; a more probable mechanism is photo-oxidation of amino acid residues of the membrane proteins. However, the lysis induced by rosoxacin is caused by photo-oxidation of cholesterol, not excluding other cellular targets.

Phototoxic potential of quinolones.

The photohaemolytic potentials of the quinolones oxolinic acid, pipemidic acid, rosoxacin, norfloxacin, ciprofloxacin and M-193324 (synthesis intermediary) were evaluated and compared with the photohaemolysis induced by nalidixic acid. Quinolones with a piperazine group in position 7 (pipemidic acid, norfloxacin and ciprofloxacin) did not induce photohaemolysis. However, oxolinic acid, rosoxacin and M-193324 produced a concentration- and oxygen-dependent photohaemolysis. Ascorbic acid, histidine and thiourea inhibited the photohaemolysis induced by oxolinic acid, rosoxacin and M-193324, suggesting a photodynamic mechanism similar to that found with nalidixic acid. In addition, deuterium oxide increased the photohaemolysis induced by photohaemolytic quinolones, indicating that this process is mediated by singlet oxygen.

Phototoxicity induced by nalidixic and oxolinic acids: decrease in cell survival of chick embryo fibroblasts and Hep-2 cells.

The phototoxic effects of nalidixic and oxolinic acids were evaluated in two types of cultured cells: chick embryo fibroblast and Hep-2 (human laryngo carcinoma cell line). In order to evaluate the phototoxicity induced by nalidixic and oxolinic acids, both cell types were irradiated for 5 min in the presence of each drug. The results showed an inverse relationship between cell survival and the concentration of the drug added to the culture medium. The concentrations of nalidixic and oxolinic acids necessary to induce a phototoxic effect were in the range of therapeutic blood levels. Both chick embryo fibroblasts and Hep-2 cells were more sensitive to the phototoxic effect induced by nalidixic acid than oxolinic acid.

Mechanism of blockade by (+)isradipine of adrenal catecholamine release.

Cat adrenal glands were perfused at a high rate with various modified Krebs solutions containing different concentrations of K+ but no Ca2+. Catecholamine release was tested by applying brief Ca2+ pulses (10 s of a solution containing 120 mM K+ and 2.5 mM Ca2+). Under polarizing conditions (10 min perfusion with 1.4 mM K+ with no Ca2+), the total catecholamines released by the Ca2+ pulse amounted to 5 micrograms; in depolarizing conditions (10 min perfusion with a solution containing 70 mM K+ but no Ca2+), secretion was somewhat less (4-4.5 micrograms). (+)Isradipine, a 1,4-dihydropyridine Ca2+ channel blocker, did not affect the secretory response under polarizing conditions at 10(-8) M; at 10(-6) M, the secretory response was halved. When present under depolarizing conditions (70 mM K+ in 0 Ca2+), (+)isradipine (10(-8) M) blocked catecholamine release by 90%. In contrast, the inorganic Ca2+ channel blocker, Co2+, inhibited secretion equally well under polarizing or depolarizing conditions. Since 45Ca2+ uptake into adrenal medullary chromaffin cells was also inhibited by (+)isradipine (10(-8) M) in a voltage-dependent manner, it seems likely that blocking effects of the drug on catecholamine release are associated with inhibition of Ca2+ entry into cells through L-type Ca2+ channels. The association of (+)isradipine to its receptor is very rapid under polarizing conditions; dissociation is very slow in depolarized cells and very rapid upon polarization of such cells. Since chromaffin cells are being depolarized during stressful situations to secrete catecholamines into the circulation, (+)isradipine is likely to bind better to dihydropyridine receptors in this state; in this manner, the ensuing blockade of adrenal secretion could serve as a protective mechanism of cardiovascular tissues against massive increases in circulating catecholamines. If this suggestion is correct this mechanism could have additional therapeutic value in the treatment of hypertensive patients with (+)isradipine.