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The rates of overweight and obesity around the world have increased in past years. The body's adipose tissue stimulates the antioxidant and oxidation imbalance capacity at the cellular level. This scenario favors an inflammatory low-grade systemic condition starting with insulin resistance, which in turn may involve diabetes mellitus type 2 and cognitive decline afterward. Neurological diseases have been correlated to senile age diseases over time. This scenario calls for a change in the incidence of obesity in the younger generation. An unhealthy dietary consumption together with sedentary habits might lead to poor gut absorption of nutrients. Several plants and foods have bioactive compounds that can reduce or inhibit radical scavengers, reactive oxygen species, and metal ion complexes that threaten the cerebral defense system. The bitter acids from hops ( Humulus lupulus L.) have been demonstrated to have promising effects on lipid and carbohydrate metabolism improvement, reducing inflammatory responses through alpha acids, beta acids, and analogs action. Therefore, the current study aimed to investigate the bioactivity of hop bitter acids in obese and lean mice. For that, a dry hop extract (DHE) was obtained by applying carbon dioxide as the fluid of supercritical extraction. Afterward, seventy-eight male mice of the C57BL/6J strain were weighed and randomly distributed into six groups of 13 animals each according to the diet offered: (NO) normolipidic diet, (NO1) normolipidic diet containing 0.35% alpha acids, (NO2) normolipidic diet containing 3.5% alpha acids, (HP) hyperlipidic diet, (HP1) hyperlipidic diet containing 0.35% alpha acids, and (HP2) hyperlipidic diet containing 3.5% alpha acids. After applying the glycemic tolerance and insulin tolerance tests, a better stabilization of glycemia levels and weight gain among those animals fed with DHE (NO2 and HP2) were observed in comparison to the obese control group (HP) ( p < 0.05). There was also an amelioration of antioxidant capacity observed by checking the enzymatic profile by SOD and an apparent mitigation of brain degeneration by checking GSK3β and p-IRS1 proteins expression ( p < 0.05). The y-maze cognitive test applied to highlight possible obesity-harmful animal brains did not indicate a statistical difference between the groups. Although the weekly dietary intake between the obese HP2 group (33.32 ± 4.11, p < 0.05) and control HP (42.3 ± 5.88, p < 0.05) was different. The bioactive compounds present in DHE have demonstrated relevant effects on glycemic control, insulin signaling, and the consequent modulatory action of the obesity-related markers with the brain's inflammatory progression.

Importance: One of the most important control points in gene regulation is RNA stability, which determines the half-life of a transcript from its transcription until its degradation. Bacteria have evolved a sophisticated multi-enzymatic complex, the RNA degradosome, which is dedicated mostly to RNA turnover. The combined activity of RNase E and the other RNA degradosome enzymes provides an efficient pipeline for the complete degradation of RNAs. The DEAD-box RNA helicases are very often found in RNA degradosomes from phylogenetically distant bacteria, confirming their importance in unwinding structured RNA for subsequent degradation. This work showed that the absence of the RNA helicase RhlB in the free-living Alphaproteobacterium Caulobacter crescentus causes important changes in gene expression and cell physiology. These are probably due, at least in part, to inefficient RNA processing by the RNA degradosome, particularly at low-temperature conditions., Competing Interests: The authors declare no conflict of interest.

Objectives: The objective of this study was to assess the effects of in situ saliva compared to in vitro human saliva, with or without mucin, on inhibiting erosion and promoting enamel rehardening., Design: Bovine enamel blocks were randomly distributed into groups (n = 23): Gsitu (human saliva in situ), Gvitro (collected human saliva) and GvitroM (collected human saliva with mucin). The enamel blocks underwent a 2-hour period for the formation of salivary pellicle, based on the assigned groups. Subsequently, they were subjected to three erosive cycles, each of them consisting of an erosive challenge (immersion in 0.65 % citric acid, pH 3.5, 1 min) and saliva exposure (immersion in situ or in vitro saliva for 2 h). Microhardness measurements were performed at each cycle, after each experimental step (erosive challenge and exposure to saliva)., Results: After the first demineralization, in vitro saliva groups presented greater hardness loss, with no statistical difference between GVitroM and GVitro. After the third erosive demineralization the in situ saliva resulted in less hardness loss compared to the first demineralization. In relation to surface hardness recovery, there was no difference among types of saliva but there was a decrease in hardness as the cycles progressed., Conclusion: Saliva groups had different behaviors between the first and third demineralization, being similar after the third cycle in terms of hardness loss. Regarding hardness recovery, all saliva promoted enamel gain, but there was a gradual decrease with the progression of the cycles., Competing Interests: Declaration of Competing Interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property., (Copyright © 2023. Published by Elsevier Ltd.)

Biochar has attracted interest due to its ability to improve soil fertility, soil carbon, and crop yield. Also, biochar can adsorb metals and render them less bioavailable. We investigated the soil availability, sequential extraction, and maize uptake of Cd, Pb, and Zn in a highly contaminated soil amended with rice straw biochar rates (0.0, 5.0, 10.0, 20.0, and 30.0 Mg ha -1 ). We hypothesized that biochar application to the soil cultivated with maize attenuates metal toxicity and mobility in slag-polluted soils near an abandoned Pb smelting plant in Brazil. Results showed that applying biochar increased the soil organic carbon, CEC, and P up to 27, 30, and 107, respectively. Plant accumulation of P and N was 104 and 32% higher than control, while aerial and root biomasses were increased by 18 and 23%. The sequential extraction showed that Pb and Zn in the original soil were retained mainly in residual fractions (94 and 87%, respectively), while Cd was mostly allocated in the organic fraction (47%). Biochar rates increased the proportion of Cd in the organic fraction to 85%, while Pb and Zn were redistributed mainly into iron oxides. The Cd, Pb, and Zn bioavailability assessed by DTPA decreased 32% in the biochar-amended soil, reducing plants' metal uptake. The maize biomass increase, metal soil bioavailability decrease, and low metal concentration in shoots driven by biochar indicate that phytoattenuation using rice straw biochar and maize cultivation could reduce risks to humans and the environment in the polluted sites of Santo Amaro., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: This two-arm, parallel-group, tooth-randomised, controlled noninferiority clinical trial aimed to compare survival rates between the sealing and restoring of cavitated occlusal carious lesions in dentine [International Caries Detection and Assessment System (ICDAS) 5] of deciduous molars using resin-modified glass-ionomer cement (RMGIC) and to assess caries progression radiographically., Materials and Methods: A total of 68 molars with ICDAS 5 occlusal lesions were randomly allocated into two groups, a sealing group (n = 31), in which RMGIC was placed directly over the carious lesion, and a restoration group (n = 37), in which a restoration with the same material was placed after selective caries removal. During the baseline and follow-up visits, dental caries was registered and caries activity was assessed according to a visuotactile criterion. At baseline, patient caries status (dmf-t) and cavity depth and extent (mesiodistal and buccolingual) were measured before RMGIC placement. An independent and blinded examiner evaluated the treated teeth using the USPHS criteria after one and two years. Standardised interproximal radiographs were taken for caries progression assessments., Results: During the follow-up period, no lesion progression was observed radiographically. After one year (n = 60; 27 sealed and 33 restored) and two years (n = 48; 23 sealed and 25 restored) of follow-up, the treatment success rates were 78.8% and 76.0% in the restoration group and 59.3% and 47.8% in the sealing group, respectively. Multivariate Cox regression showed that lesions smaller than 2 mm in the mesiodistal extent were less prone to fail after one year (p = 0.03). However, survival curves (log-rank test) were statistically significantly different only after two years (p < 0.001)., Conclusions: Sealing ICDAS 5 occlusal lesions of deciduous molars using RMGIC achieved lower survival rates than restorations. Both sealing and restoration effectively arrested caries progression for two years. Clinical relevance Sealing dentine carious lesions can be effective for treating lesions involving the inner and outer half of the dentine. Ultraconservative treatments can arrest carious lesions presenting obvious cavitation in primary molars., Trial Registration: ReBEC Register no. RBR-225n35., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The aim of this study was to compare the acquired enamel pellicle protein profile of professional wine tasters with mild and moderate erosive tooth wear. Twelve professional wine tasters participated (3 from a low tooth wear group; 9 from a high tooth wear group). Acquired enamel pellicle samples were collected and processed for proteomic analysis (nLC-ESI-MS/MS). The acquired enamel pellicle proteomic profile was different between the groups. The proteins found exclusively in the low tooth wear group were histatins 1 and 3 and mucins 7 and 21. When comparing the wear groups, proteins with higher levels in the low tooth wear group included neutrophil defensins (1 and 3), lysozyme C, lysozyme, myeloperoxidase, and squalene monooxygenase. In conclusion, the findings indicate that the proteins found at higher levels in the low tooth wear group and proteins exclusively found in the low tooth wear group might be protective and, therefore, could be good candidates for further studies regarding their potential to be added to dental products to protect professional wine tasters from extrinsic erosive tooth wear., (© 2021 European Journal of Oral Sciences.)

This study evaluated the effect of lacosamide (LCM) on biochemical and mitochondrial parameters after PTZ kindling in mice. Male mice were treated on alternative days for a period of 11 days with LCM (20, 30, or 40 mg/kg), saline, or diazepam (2 mg/kg), before PTZ administration (50 mg/kg). The hippocampi were collected to evaluate free radicals, the activities of superoxide dismutase (SOD), catalase (CAT), and the mitochondrial complexes I-III, II, and II-III, as well as Bcl-2 and cyclo-oxygenase-2 (COX-2) expressions. Hippocampi, blood, and bone marrow were collected for genotoxic and mutagenic evaluations. LCM 40 mg/kg increased latency and decreased percentage of seizures, only on the 3rd day of observation. The dose of 30 mg/kg only showed positive effects on the percentage of seizures on the 2nd day of observation. LCM decreased free radicals and SOD activity and the dose of 40 mg/kg were able to increase CAT activity. LCM 30 and 40 mg/kg improved the enzymatic mitochondrial activity of the complex I-III and LCM 30 mg/kg improved the activity of the complex II. In the comet assay, the damage induced by PTZ administration was reduced by LCM 20 and 30 mg/kg. The dose of 20 mg/kg increased COX-2 expression while the highest dose used, 40 mg/kg, was able to reduce this expression when compared to the group treated with LCM 20 mg/kg. Although LCM did not produce the antiepileptogenic effect in vivo, it showed the neuroprotective effect against oxidative stress, bioenergetic dysfunction, and DNA damage induced by the repeated PTZ administration., (© 2020 Société Française de Pharmacologie et de Thérapeutique.)

Aims: To study the correlation between the workload of intensive care nursing teams and the sociodemographic, anthropometric and clinical characteristics of patients in critical condition in a Portuguese Intensive Care Unit (ICU) during a 5-year period., Background: Currently, indices of nursing workload quantification are one of the resources used for planning and evaluating ICUs. Evidence shows that there are several factors related to critical patients and their hospitalisation which potentially influence the nursing workload., Design: Retrospective cohort analysis of a health record database from adult patients admitted to a Portuguese ICU between 1 January 2015-31 December 2019., Methods: Simplified Therapeutic Intervention Scoring System (TISS-28) scores of 730 adult patients. Three TISS-28 assessments were considered: first assessment, last assessment and average. The STROBE guidelines were used in reporting this study., Results: The TISS-28 has an average of 34.2 ± 6.9 points at admission, which is considered a high nursing workload. A somewhat lower result was found for the discharge and average assessments. It shows that basic activities accounted for the highest percentage of time spent (38.0%), followed by the cardiovascular support category (26.5%). The TISS-28 shows consistent results throughout the study period, despite a small trend reduction in the last 2 years., Conclusions: Lower workloads were found for age ≤44 years and with a shorter length of stay. Higher workload was more probable in patients classified in Cullen Class IV (OR = 2.5) and with a normal to higher weight percentile (OR = 1.9 and 1.5, respectively)., Relevance to Clinical Practice: Knowledge of the factors influencing the nursing workload facilitates the implementation of rules to improve performance in nursing interventions, based on the redefinition of care priorities, increased productivity, human resources management and reduction of additional costs to the organisation, related to possible adverse events, among others., (© 2020 John Wiley & Sons Ltd.)

Background: The aim was to evaluate whether aspartame regular mouthwash prior to erosive challenges with citric or hydrochloric acids would be able to prevent erosive enamel wear., Material and Methods: This randomized, single blind in situ study was conducted with 3 crossover phases of 5 days. Polished bovine enamel blocks (n=252) were randomly divided among 6 groups/ 3 phases/ 21 volunteers. The groups under study were: aspartame solution (0.024% of aspartame in deionized water - experimental group), deionized water (negative-control) and stannous-containing solution (Elmex® Erosion Protection Dental Rinse; positive-control); subjected to erosion on citric acid or hydrochloric acid. Four times per day the volunteers rinsed the intraoral appliance with the respective solutions ( in situ ) prior to immersion of half of the appliance in 0.05M citric acid and the other half in 0.01M hydrochloric acid for 120 seconds (extraoral). The response variable was enamel loss by profilometry. Data were analyzed by ANOVA and Tukey's test ( p <0.05)., Results: No difference on enamel loss was found between aspartame solution and deionized water. Stannous-solution resulted in less enamel loss compared to deionized water. Hydrochloric acid resulted in higher enamel loss than citric acid., Conclusions: In this model, aspartame was not able to prevent erosive tooth wear against citric or hydrochloric acids. Key words: Dental erosion, aspartame, stannous fluoride, citric acid, hydrochloric acid., Competing Interests: Conflicts of interest The authors declare that they have no conflict of interest., (Copyright: © 2020 Medicina Oral S.L.)

The aim of this study was to investigate the effects of high hydrostatic pressure (HHP) on the inactivation of Lactobacillus fructivorans, on the inactivation of Alicyclobacillus acidoterrestris spores and on the extraction of anthocyanins and total phenolics from açaí pulp. The tested conditions comprised pressures of 400-600 MPa, treatment times of 5-15 min, and temperatures of 25 °C and 65 °C. Results were compared to those of conventional thermal treatments (85 °C/1 min). Regarding A. acidoterrestris spores, applying HHP for 13.5 min, resulted in a value of four-decimal reduction. L. fructivorans presented considerable sensitivity to HHP treatment, achieving inactivation rates above 6.7 log cycles at process conditions at 600 MPa and 65 °C for 5 min. All samples of açaí pulp processed showed absence of thermotolerant coliforms during the 28 days of refrigerated storage (shelf life study). The açaí pulps processed by HHP (600 MPa/5 min/25 °C) had anthocyanin extraction increased by 37% on average. In contrast, conventional thermal treatment reduced anthocyanin content by 16.3%. For phenolic compounds, the process at 600 MPa/5 min/65 °C increases extraction by 10.25%. A combination of HHP treatment and moderate heat (65 °C) was shown to be an alternative to thermal pasteurization, leading to microbiologically safe products while preserving functional compounds., Competing Interests: Declaration of Competing Interest The authors declared that there is no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Diabetic neuropathy is the most prevalent complication associated with diabetes mellitus (DM). The superior cervical ganglion (SCG) is an important sympathetic component of the autonomic nervous system. We investigated the changes in cellular electrophysiological properties and on Na + K + -ATPase activity of SCG neurons of rats with DM induced by streptozotocin (STZ). Three types of action potentials (AP) firing pattern were observed in response to a long (1 s) depolarizing pulse. Whilst some neurons fired a single AP (single firing phasic, SFP), others fired few APs (multiple firing phasic, MFP). A third type fired APs during more than 80% of the stimulus duration (tonic-like, TL). The occurrence of SFP, MFP and TL was 84.5, 13.8, and 1.7%, respectively. SFP and MFP differed significantly in their membrane input resistance (R in ). At the end of the 4th week of its time course, DM differently affected most types of neurons: DM induced depolarization of resting membrane potential (RMP), decreased AP amplitude in SFP, and decreased R in in MFP. DM decreased spike after-hyperpolarization amplitude in MFP and the duration in SFP. Based on the RMP depolarization, we investigated the Na + K + -ATPase action and observed that DM caused a significant decrease in Na + K + -ATPase activity of SCG. In conclusion, we have demonstrated that DM affects several parameters of SCG physiology in a manner likely to have pathophysiological relevance., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Erosive tooth wear often leads to surface loss requiring restoration of primary teeth with adhesive materials. The purpose of this study was to evaluate the microshear bond strength (μSBS) of different adhesive systems to sound and eroded primary enamel and dentin surfaces. Methods: Half of the samples underwent erosion (immersion in Coca-Cola for one minute five times over five days) and abrasion cycles (brushing with an electric toothbrush, fluoride toothpaste slurry, for one minute at 250 g load). Samples were divided into adhesive groups ( n equals 12): Adper Single Bond 2 (etch-and-rinse); Single Bond Universal (self-etch); Optibond FL (etch-and-rinse with fluoride); and Bond-Force (self-etch with fluoride). Resin composite was bonded on sample surfaces and subjected to μSBS test. Data were analyzed by two-way analysis of variance, Tukey test, and chi-square test ( P <0.05). The failure mode was determined using a stereomicroscope under 20X magnification. Results: No difference was found between the eroded and sound enamel and dentin ( P ≥ 0.05) on μSBS test. The failure mode evaluation showed significant differences between sound and eroded dentin ( P =0.003) but no difference among the adhesives ( P =0.177). Conclusions: Micro-shear bond strength in primary enamel and dentin was not affected by erosion/abrasion or type of adhesive system.

The monoterpenoid terpinen-4-ol (4TERP) is known to inhibit cell excitability, has low toxicity and important pharmacological activities, which are likely related to neural excitability, such as anti-inflammatory, antiepileptic and antinociceptive effects. However, the pharmacological characteristics and mechanisms underlying the effects of 4TERP on blockade of neural action potential are not completely elucidated. Since Na + current (I Na ) through voltage-dependent Na + channels (Na V ) is a major mechanism for excitability, the present study investigated the pharmacological characteristics and mechanisms of the action of 4TERP on I Na through Na V . For this aim, dissociated small neurons of dorsal root ganglia of adult rats were used for whole cell patch-clamp recordings. 4TERP concentration-dependently inhibits I Na (IC 50 0.8 ± 0.3 mM; pharmacological efficacy 42.89 ± 5.54%). 4TERP interfered with I Na through a mechanism with various components, which includes predominantly channel pore block and sensitivity to frequency of use. In presence of 4TERP (3 mM), decreasing stimulation from 5 Hz to very low frequency (75 s of quiescence previously to stimulation) induced I Na decrease to 65.17 ± 5.86% of control. 4TERP also altered (left shift) voltage sensitivity of the steady state activation of Na V . Data are discussed aiming to interpret the importance of blockade of I Na through Na V as participant of 4TERP-induced inhibition of membrane excitability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: The aim of this study was to investigate the effect of a dentifrice that contains calcium silicate, sodium phosphate, and fluoride on erosive-abrasive enamel wear., Material and Methods: This randomized, single-blind in situ/ex vivo study was conducted with four crossover phases of 5 days (one group tested per phase). Bovine enamel blocks (n = 256) were allocated to 16 volunteers and 8 groups. The groups under study were test dentifrice, with calcium silicate, sodium phosphate, and 1450 ppm sodium monofluorophosphate; tin dentifrice, with 3500 ppm stannous chloride, 700 ppm amine fluoride, and 700 ppm sodium fluoride; conventional dentifrice, with 1450 ppm sodium monofluorophosphate; and control (deionized water). Half of the enamel blocks were subjected to erosion and the other half to erosion plus abrasion. The daily extraoral protocol consisted in four citric acid exposures (2 min) and two applications of dentifrice slurry on all blocks for 30 s; after, half of the blocks were brushed for 15 s. The response variable was enamel loss. Data were analyzed by two-way ANOVA and Fisher's test (p < 0.05)., Results: For erosion, the test dentifrice promoted less enamel loss than water (4.7 ± 3.1 and 5.8 ± 2.5 μm, respectively, p < 0.05), and did not differ from tin (4.8 ± 2.5 μm) and conventional (4.8 ± 1.4 μm) dentifrices (p > 0.05). However, the test dentifrice (7.7 ± 3.8 μm) promoted higher wear after erosive plus abrasive procedures than tin (5.4 ± 1.5 μm) and conventional (6.2 ± 1.7 μm, p < 0.05) dentifrices, and did not differ from water (6.9 ± 2.0 μm)., Conclusions: The investigated dentifrice reduced enamel loss against acid challenge but had no effect against acid and brushing challenge., Clinical Relevance: Little is known regarding the preventive effect of dentifrices indicated for dental erosion. The tested anti-erosive dentifrice was unable to protect enamel when erosion was associated to toothbrushing abrasion.

Melatonin is a neurohormone produced and secreted at night by pineal gland. Many effects of melatonin have already been described, for example: Activation of potassium channels in the suprachiasmatic nucleus and inhibition of excitability of a sub-population of neurons of the dorsal root ganglia (DRG). The DRG is described as a structure with several neuronal populations. One classification, based on the repolarizing phase of the action potential (AP), divides DRG neurons into two types: Without (N 0 ) and with (N inf ) inflection on the repolarization phase of the action potential. We have previously demonstrated that melatonin inhibits excitability in N 0 neurons, and in the present work, we aimed to investigate the melatonin effects on the other neurons (N inf ) of the DRG neuronal population. This investigation was done using sharp microelectrode technique in the current clamp mode. Melatonin (0.01-1000.0 nM) showed inhibitory activity on neuronal excitability, which can be observed by the blockade of the AP and by the increase in rheobase. However, we observed that, while some neurons were sensitive to melatonin effect on excitability (excitability melatonin sensitive-EMS), other neurons were not sensitive to melatonin effect on excitability (excitability melatonin not sensitive-EMNS). Concerning the passive electrophysiological properties of the neurons, melatonin caused a hyperpolarization of the resting membrane potential in both cell types. Regarding the input resistance (R in ), melatonin did not change this parameter in the EMS cells, but increased its values in the EMNS cells. Melatonin also altered several AP parameters in EMS cells, the most conspicuously changed was the (dV/dt) max of AP depolarization, which is in coherence with melatonin effects on excitability. Otherwise, in EMNS cells, melatonin (0.1-1000.0 nM) induced no alteration of (dV/dt) max of AP depolarization. Thus, taking these data together, and the data of previous publication on melatonin effect on N 0 neurons shows that this substance has a greater pharmacological potency on N inf neurons. We suggest that melatonin has important physiological function related to N inf neurons and this is likely to bear a potential relevant therapeutic use, since N inf neurons are related to nociception.

In C. crescentus , iron metabolism is mainly controlled by the transcription factor Fur (ferric uptake regulator). Iron-bound Fur represses genes related to iron uptake and can directly activate the expression of genes for iron-containing proteins. In this work, we used total RNA sequencing (RNA-seq) of wild type C. crescentus growing in minimal medium under iron limitation and a fur mutant strain to expand the known Fur regulon, and to identify novel iron-regulated genes. The RNA-seq of cultures treated with the iron chelator 2-2-dypiridyl (DP) allowed identifying 256 upregulated genes and 236 downregulated genes, being 176 and 204 newly identified, respectively. Sixteen transcription factors and seven sRNAs were upregulated in iron limitation, suggesting that the response to low iron triggers a complex regulatory network. Notably, lexA along with most of its target genes were upregulated, suggesting that DP treatment caused DNA damage, and the SOS DNA repair response was activated in a RecA-dependent manner, as confirmed by RT-qPCR. Fluorescence microscopy assays using an oxidation-sensitive dye showed that wild type cells in iron limitation and the fur mutant were under endogenous oxidative stress, and a direct measurement of cellular H 2 O 2 showed that cells in iron-limited media present a higher amount of endogenous H 2 O 2 . A mutagenesis assay using the rpoB gene as a reporter showed that iron limitation led to an increase in the mutagenesis rate. These results showed that iron deficiency causes C. crescentus cells to suffer oxidative stress and to activate the SOS response, indicating an increase in DNA damage.

Melatonin, a powerful antioxidant, participates in the regulation of important physiological and pathological processes. We investigated the actions of melatonin on neuronal excitability of intact dorsal root ganglions (DRG) from rats using intracellular recording techniques in current clamps. Melatonin blocked the generation of action potentials in a concentration-dependent manner. Bath applied melatonin (1.0-1000.0 nM) hyperpolarized the resting membrane potential, and increased the input resistance and rheobase. Melatonin also altered the active electrophysiological properties of the action potential, amplitude and maximum descendant inclination, in a statistically significant way. In order to provide evidence on the mechanism of action of melatonin in the DRG, quantitative PCR (qPCR) was performed. Analyses were performed for melatonin membrane receptors, MT 1 and MT 2 , and it was observed that the DRG expresses MT 1 receptors. In addition, we noted that the melatonin-induced effects were blocked in the presence of luzindole, a melatonin receptor antagonist. The minimal effective concentrations of melatonin (10.0 nM) and the blockade of effects caused by luzindole suggest that the effects of melatonin are hormonal, and are induced when it binds to MT 1 receptors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Here in, we report the preparation and evaluation of four 3-hydroxy-piperidine-N-benzyl-aryl-acylhydrazone derivatives (6a-d) for their potential antinociceptive activity. In the tail flick test, compounds 6a and 6d exhibited a significant increase in the latency time of the animals, in comparison to the control group. These two compounds also showed a significant increase in the nociceptive threshold from 1 to 6 h after treatment in the CCI neuropathic pain model. In both cases, the antinociceptive activity was blocked by naloxone, suggesting an opioid mechanism of action, but without sedative or motor coordination effects., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Todos os Santos (All Saints) Bay area, NE-Brazil, is known for one of the most important cases of urban lead (Pb) contamination in the world. The main objective of this work was to assess and interpret the spatial distribution of As, Cd, Hg, Pb, and Zn in "background" soils of this environmentally impacted bay area, using a combination of geostatistical and multivariate analytical methods to distinguish between natural and anthropogenic sources of those metal(oid)s in soils. We collected 114 topsoil samples (0.0-0.2 m depth) from 38 sites. The median values for trace metal concentrations in soils (mg kg -1 ) followed the order Pb (33.9) > Zn (8.8) > As (1.2) > Cd (0.2) > Hg (0.07), clearly reflecting a Pb-contamination issue. Principal component analysis linked Cd, Pb, and Zn to the same factor (F1), chiefly corroborating their anthropogenic origin; yet, both Pb and Zn are also influenced by natural lithogenic sources. Arsenic and Hg concentrations (F2) are likely related to the natural component alone; their parent material (igneous-metamorphic rocks) seemingly confirm this hypothesis. The heterogeneity of sources and the complexity of the spatial distribution of metals in large areas such as the Todos os Santos Bay warrant, the importance of multivariate and geostatistical analyses in the interpretation of environmental data.

AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

PDI (PDIA1) and ERp57 (PDIA3), members of the PDI family and of the thioredoxin (Trx) superfamily, are multifunctional proteins with wide physiological roles and have been implicated in several pathologies. Importantly, they are both involved in the MHC class I antigen presentation pathway. This paper reports the isolation and characterization of full cDNA and genomic clones from sea bass (Dicentrarchus labrax, L.) PDI (Dila-PDI) and ERp57 (Dila-ERp57). The genes are ~12.4 and ~7.1 kb long, originating 2155 and 2173 bp transcripts and encoding 497 and 484 amino acids mature proteins, for Dila-PDI and -ERp57, respectively. The PDI gene consists of eleven exons and ERp57 of thirteen. As described in other species, both molecules are composed of four Trx-like domains (abb'a') followed by a C-terminal tail, retaining two CGHC active sites and an ER-signalling sequence, suggestive of a conserved function. Additionally, three-dimensional homology models further support Dila-PDI and Dila-ERp57 as orthologs of mammalian PDI and ERp57, respectively. Finally, high similarity is observed to their vertebrate counterparts (>69% identity), especially among the few ones from closely related teleosts (>79% identity). Hence, these results provide relevant primary data and will enable further studies to clarify the roles of PDI and ERp57 in European sea bass immunity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Mammalian calreticulin (CRT) is a key molecular chaperone and regulator of Ca(2+) homeostasis in endoplasmic reticulum (ER), also being implicated in a variety of physiological/pathological processes outside the ER. Importantly, it is involved in assembly of MHC class I molecules. In this work, sea bass (Dicentrarchus labrax) CRT (Dila-CRT) gene and cDNA have been isolated and characterized. The mature protein retains two conserved motifs, three structural/functional domains (N, P and C), three type 1 and 2 motifs repeated in tandem, a conserved pair of cysteines and ER-retention motif. It is a single-copy gene composed of 9 exons. Dila-CRT three-dimensional homology models are consistent with the structural features described for mammalian molecules. Together, these results are supportive of a highly conserved structure of CRT through evolution. Moreover, the present data provides information that will allow further studies on sea bass CRT involvement in immunity and in particular class I antigen presentation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this work, the gene and cDNA of sea bass (Dicentrarchus labrax) β2-microglobulin (Dila-β2m) and several cDNAs of MHC class I heavy chain (Dila-UA) were characterized. While Dila-β2m is single-copy, numerous Dila-UA transcripts were identified per individual with variability at the peptide-binding domain (PBD), but also with unexpected diversity from the connective peptide (CP) through the 3' untranslated region (UTR). Phylogenetic analysis segregates Dila-β2m and Dila-UA into each subfamily cluster, placing them in the fish class and branching Dila-MHC-I with lineage U. The α1 domains resemble those of the recently proposed L1 trans-species lineage. Although no Dila-specific α1, α2 or α3 sub-lineages could be observed, two highly distinct sub-lineages were identified at the CP/TM/CYT regions. The three-dimensional homology model of sea bass MHC-I complex is consistent with other characterized vertebrate structures. Furthermore, basal tissue-specific expression profiles were determined for both molecules, and expression of β2m was evaluated after poly I:C stimulus. Results suggest these molecules are orthologues of other β2m and teleost classical MHC-I and their basic structure is evolutionarily conserved, providing relevant information for further studies on antigen presentation in this fish species., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

AIP56 (apoptosis-inducing protein of 56 kDa) is a major virulence factor of Photobacterium damselae piscicida (Phdp), a Gram-negative pathogen that causes septicemic infections, which are among the most threatening diseases in mariculture. The toxin triggers apoptosis of host macrophages and neutrophils through a process that, in vivo, culminates with secondary necrosis of the apoptotic cells contributing to the necrotic lesions observed in the diseased animals. Here, we show that AIP56 is a NF-κB p65-cleaving zinc-metalloprotease whose catalytic activity is required for the apoptogenic effect. Most of the bacterial effectors known to target NF-κB are type III secreted effectors. In contrast, we demonstrate that AIP56 is an A-B toxin capable of acting at distance, without requiring contact of the bacteria with the target cell. We also show that the N-terminal domain cleaves NF-κB at the Cys(39)-Glu(40) peptide bond and that the C-terminal domain is involved in binding and internalization into the cytosol.

Three-dimensional gait analysis (3DGA) is an important element in the quantitative evaluation of gait in subjects with Parkinson's disease (PD). Indexes, such as the Gait Deviation Index (GDI), have recently been proposed as a summary measure of gait. The aim of the present study was to investigate the effectiveness of the GDI and spatiotemporal variables in the quantification of changes in gait during a dual-task (DT) exercise. Fourteen patients with idiopathic PD and nine healthy subjects (CG) participated in the study. All subjects walked under two conditions: free walking and DT walking. The GDI was computed from the 3DGA data. The results show gait impairment during DT, a significant difference between groups regarding GDI and an interaction effect involving the group, side and task factors. The CG and PDG were different independent of interference and side, but interference was only different for the PDG group. The results also demonstrate that the GDI should be an appropriate outcome measure for the evaluation of the effects of DT on patients with Parkinson's disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Interleukine-1β (IL-1β) is the most studied pro-inflammatory cytokine, playing a central role in the generation of systemic and local responses to infection, injury, and immunological challenges. In mammals, IL-1β is synthesized as an inactive 31 kDa precursor that is cleaved by caspase-1 generating a 17.5 kDa secreted active mature form. The caspase-1 cleavage site strictly conserved in all mammalian IL-1β sequences is absent in IL-1β sequences reported for non-mammalian vertebrates. Recently, fish caspase-1 orthologues have been identified in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) but very little is known regarding their processing and activity. In this work it is shown that sea bass caspase-1 auto-processing is similar to that of the human enzyme, resulting in active p24/p10 and p20/p10 heterodimers. Moreover, the presence of alternatively spliced variants of caspase-1 in sea bass is reported. The existence of caspase-1 isoforms in fish and in mammals suggests that they have been evolutionarily maintained and therefore are likely to play a regulatory role in the inflammatory response, as shown for other caspases. Finally, it is shown that sea bass and avian IL-1β are specifically cleaved by caspase-1 at different but phylogenetically conserved aspartates, distinct from the cleavage site of mammalian IL-1β.

Mammalian tapasin (TPN) is a key member of the major histocompatibility complex (MHC) class I antigen presentation pathway, being part of the multi-protein complex called the peptide loading complex (PLC). Several studies describe its important roles in stabilizing empty MHC class I complexes, facilitating peptide loading and editing the repertoire of bound peptides, with impact on CD8(+) T cell immune responses. In this work, the gene and cDNA of the sea bass (Dicentrarchus labrax) glycoprotein TPN have been isolated and characterized. The coding sequence has a 1329 bp ORF encoding a 442-residue precursor protein with a predicted 24-amino acid leader peptide, generating a 418-amino acid mature form that retains a conserved N-glycosylation site, three conserved mammalian tapasin motifs, two Ig superfamily domains, a transmembrane domain and an ER-retention di-lysine motif at the C-terminus, suggestive of a function similar to mammalian tapasins. Similar to the human counterpart, the sea bass TPN gene comprises 8 exons, some of which correspond to separate functional domains of the protein. A three-dimensional homology model of sea bass tapasin was calculated and is consistent with the structural features described for the human molecule. Together, these results support the concept that the basic structure of TPN has been maintained through evolution. Moreover, the present data provides information that will allow further studies on cell-mediated immunity and class I antigen presentation pathway in particular, in this important fish species., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The transporters associated with antigen processing (TAP), play an important role in the MHC class I antigen presentation pathway. In this work, sea bass (Dicentrarchus labrax) TAP1 and TAP2 genes and transcripts were isolated and characterized. Only the TAP2 gene is structurally similar to its human orthologue. As other TAP molecules, sea bass TAP1 and TAP2 are formed by one N-terminal accessory domain, one core membrane-spanning domain and one canonical C-terminal nucleotide-binding domain. Homology modelling of the sea bass TAP dimer predicts that its quaternary structure is in accordance with that of other ABC transporters. Phylogenetic analysis segregates sea bass TAP1 and TAP2 into each subfamily cluster of transporters, placing them in the fish class and suggesting that the basic structure of these transport-associated proteins is evolutionarily conserved. Furthermore, the present data provides information that will enable more studies on the class I antigen presentation pathway in this important fish species., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

It has been previously shown that the exotoxin of the important fish pathogen Photobacterium damselae ssp. piscicida is a key pathogenicity factor and is responsible for the extensive systemic apoptosis of macrophages and neutrophils seen in acute fish photobacteriosis. The focus of the present study was to further characterize the AIP56-induced apoptosis of sea bass professional phagocytes by assessing the involvement of caspases, mitochondria and oxidative stress. The resulting data indicate that the apoptotic response in peritoneal macrophages and neutrophils treated ex vivo with AIP56 involves activation of caspase-8, -9 and -3, and mitochondria as shown by loss of mitochondrial membrane potential, release of cytochrome c and over-production of ROS. These results together with previous data from this laboratory suggest that both the extrinsic and intrinsic apoptotic pathways are involved in the AIP56-induced phagocyte apoptosis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This study aims to identify the conflicts found daily throughout the development of the specialization course Process of Change in Superior Education and Health Services. This descriptive qualitative study is a case study carried out at a private University Center in the State of Rio de Janeiro, Brazil. The subjects were 18 students from the specialization course, who spontaneously decided to participate in the study The data was collected in April 2009 and analyzed and described in five thematic units that describe the focus of conflicts in the world of the work: Integration among Education, Work and Citizenship; Team work; Permanent education, Social responsibility; Establishment of the care line. The intersection between the academic field and health services produces tensions and conflicts which are latent most of the time, but are never evident.

Caspase-8 is an initiator caspase that plays a crucial role in some cases of apoptosis by extrinsic and intrinsic pathways. Caspase-8 structure and function have been extensively studied in mammals, but in fish the characterization of that initiator caspase is still scarce. In this work, the sea bass counterpart of mammalian caspase-8 was sequenced and characterized, and its involvement in the apoptogenic activity of a toxin from a fish pathogen was assessed. A 2472 bp cDNA of sea bass caspase-8 was obtained, consisting of 1455 bp open reading frame coding for 484 amino acids and with a predicted molecular weight of 55.2 kDa. The sea bass caspase-8 gene has 6639 bp and is organized in 11 introns and 12 exons. Several distinctive features of sea bass caspase-8 were identified, which include two death effector domains, the caspase family domains p20 and p10, the caspase-8 active-site pentapeptide and potential aspartic acid cleavage sites. The sea bass caspase-8 sequence revealed a significant degree of similarity to corresponding sequences from several vertebrate taxonomic groups. A low expression of sea bass caspase-8 was detected in various tissues of non-stimulated sea bass. Furthermore, it is shown that stimulation of sea bass with mid-exponential phase culture supernatants from Photobacterium damselae ssp. piscicida (Phdp), known to induce selective apoptosis of macrophages and neutrophils, resulted in an increased expression of caspase-8 in the spleen, one of the main affected organs by Phdp infection., (2010 Elsevier Ltd. All rights reserved.)

Photobacterium damselae subsp. piscicida (Phdp) is a Gram-negative pathogen agent of an important fish septicemia. The key virulence factor of Phdp is the plasmid-encoded exotoxin AIP56, which is secreted by exponentially growing pathogenic strains. AIP56 has 520 amino acids including an N-terminal cleavable signal peptide of 23 amino acid residues, two cysteine residues and a zinc-binding region signature HEXXH that is typical of most zinc metallopeptidases. AIP56 induces in vitro and in vivo selective apoptosis of fish macrophages and neutrophils through a caspase-3 dependent mechanism that also involves caspase-8 and -9. In vivo, the AIP56-induced phagocyte apoptosis progresses to secondary necrosis with release of cytotoxic phagocyte molecules including neutrophil elastase. Fish injected with recombinant AIP56 die with a pathology similar to that seen in the natural infection.

Recent in vitro studies have demonstrated that copper may induce apoptosis triggering the activation of caspase-3, a central effector of apoptotic cell death. However, the precise mechanism of copper-induced apoptosis is still unclear, even less so in Oreochromis niloticus where no caspase genes have been reported so far. This study aimed to assess the in vivo role of copper in apoptosis induction on O. niloticus gill, simultaneously contributing to elucidate the mechanism of copper-induced apoptosis. Caspase-3 gene was partially sequenced and, after in vivo exposures to 40 and 400 microgL(-1) of copper, its mRNA expression was evaluated by real-time PCR. Apoptosis was also evaluated by TUNEL assay and cell proliferation identified using an antibody against proliferating cell nuclear antigen (PCNA). The copper concentrations used did not induce the upregulation of caspase-3 gene in O. niloticus gill. In addition, in the gills of fish exposed to copper there was no increase in the estimated relative volume of apoptotic cells, indicating that neither the caspase-3-dependent or caspase-independent apoptotic pathways were induced. On the other hand, the increase in the volumetric density of epithelial proliferating cells suggests a concentration-dependent repair response.

In metazoans apoptosis is a major physiological process of cell elimination during development and in tissue homeostasis and can be involved in pathological situations. In vitro, apoptosis proceeds through an execution phase during which cell dismantling is initiated, with or without fragmentation into apoptotic bodies, but with maintenance of a near-to-intact cytoplasmic membrane, followed by a transition to a necrotic cell elimination traditionally called "secondary necrosis". Secondary necrosis involves activation of self-hydrolytic enzymes, and swelling of the cell or of the apoptotic bodies, generalized and irreparable damage to the cytoplasmic membrane, and culminates with cell disruption. In vivo, under normal conditions, the elimination of apoptosing cells or apoptotic bodies is by removal through engulfment by scavengers prompted by the exposure of engulfment signals during the execution phase of apoptosis; if this removal fails progression to secondary necrosis ensues as in the in vitro situation. In vivo secondary necrosis occurs when massive apoptosis overwhelms the available scavenging capacity, or when the scavenger mechanism is directly impaired, and may result in leakage of the cell contents with induction of tissue injury and inflammatory and autoimmune responses. Several disorders where secondary necrosis has been implicated as a pathogenic mechanism will be reviewed.

Apoptosis is a genetically controlled and evolutionarily conserved form of active cell death, albeit with an increase in complexity with continuing development. A high conservation at the functional and molecular level has been described between the players of the apoptotic machinery in invertebrates (Caenorhabditis elegans and Drosophila) and mammals. However, fish represent an excellent and advantageous model for the study of vertebrate development and disease, bridging the gap between the C. elegans/Drosophila and mouse/human models. Moreover, contrary to C. elegans and Drosophila, fish can be used for studying the development and function of vertebrate-specific organs and have a fully developed immune system similar to that of mammals. Last but not less important, both the environment and human health will obviously gain by using the knowledge generated through the use of fish models, for developing better prophylactic and therapeutic measures with impact on the aquaculture industry. In the present article, structural and functional data on the most important apoptosis related molecules, namely death-receptor, Bcl-2 and caspase families, and mechanisms are reviewed. The data point to the existence in fish of apoptotic pathways equivalent to those of mammals, making fish useful animal models for studying apoptosis, which may have great applicability for the advance of the knowledge on the role of apoptotic cell death in human apoptosis-related disorders as well as in pharmaceutical design.

The relevance of fish research has been rising due to the expansion of aquaculture and to the increasing use of fish as replacements for mammals in the study of human physiological and pathological issues. Fish have much smaller genomes compared to mammals, and zebrafish, fugu, medaka and spotted green puffer fish have the sequence of their genomes completed or near completion. Fish have several of the virtues of Drosophila melanogaster and Caenorhabditis elegans for apoptosis research, but offer additional advantages because they are vertebrates and have a developed immune system and apoptotic pathways similar to those of mammals. Many phenotypes in the zebrafish resemble human diseases and this fish has been increasingly used in pharmaceutical design of apoptosis modulating drugs. The roles of microRNAs, bcl-2, p53, insulin-like growth factor-binding protein-3, and cellular apoptosis susceptibility (CAS) and c-Myc genes (involved in the interaction apoptosis/cancer), and Abeta peptides, presenilin enhancer 2, cyclin-dependent kinase 5 and tau (factors with relevant roles in apoptosis-associated human neurodegenerative disorders), have also been successfully investigated in fish models. Results of research with fish that have advanced the knowledge on the participation of apoptosis in viral infections and of apoptosis and secondary necrosis in bacterial infections are also reviewed. It is expectable that the use of fish for research on apoptosis-related issues relevant for human physiology and pathology and for the design of apoptosis-modulating drugs will continue to increase.

In the search for pro-inflammatory genes in sea bass a TNF-alpha gene was cloned and sequenced. The sea bass TNF-alpha (sbTNF-alpha) putative protein conserves the TNF-alpha family signature, as well as the two cysteines usually involved in the formation of a disulfide bond. The mouse TNF-alpha Thr-Leu cleavage sequence and a potential transmembrane domain were also found, suggesting that sbTNF-alpha exists as two forms: a approximately 28 kDa membrane-bound form and a approximately 18.4 kDa soluble protein. The single copy sbTNF-alpha gene contains a four exon-three intron structure similar to other known TNF-alpha genes. Homology modeling of sbTNF-alpha is compatible with the trimeric quaternary architecture of its mammalian counterparts. SbTNF-alpha is constitutively expressed in several unstimulated tissues, and was not up-regulated in the spleen and head-kidney, in response to UV-killed Photobacterium damselae subsp. piscicida. However, an increase of sbTNF-alpha expression was detected in the head-kidney during an experimental infection using the same pathogen.

The present manuscript reports for the first time the sequencing and characterisation of sea bass (sb) MHCII alpha and beta chains and Ii chain cDNAs as well as their expression analysis under resting state. 3D homology modelling, using crystal structures from mammalian orthologues, has been used to illustrate and support putative structural homologies of the sea bass counterparts. The sbIi cDNA consists of 96 bp of 5'-UTR, a 843 bp open reading frame (ORF) and 899 bp of 3'-UTR including a canonical polyadenylation signal 16 nucleotides before the polyadenylation tail. The ORF was translated into a 280 amino acid sequence, in which all characteristic domains found in the Ii p41 human form could be identified, including the cytoplasmic N-terminus domain, the transmembrane (TM) region, the CLIP domain, the trimerization domain and the thyroglobulin (Tg) type I domain. The trimerization and Tg domains of sbIi were successfully modelled using the human counterparts as templates. Four different sequences of each class II alpha and beta MHCII were obtained from a single fish, apparently not derived from a single locus. All the characteristic features of the MHCII chain structure could be identified in the predicted ORF of sea bass alpha and beta sequences, consisting of leader peptide (LP), alpha1/beta1 and alpha2/beta2 domains, connecting peptide and TM and cytoplasmic regions. Furthermore, independently of the HLA-DR crystal structure used as template in homology modelling, a similar predicted 3D structure and trimeric quaternary architecture was obtained for sbMHC, with major deviations occurring only within the sea bass MHCII alpha1 domain.

Detachment-induced apoptosis of enterocytes (anoikis) has not been investigated in vivo. Here we describe anoikis of fish enterocytes following detachment in a septicemia by Photobacterium damselae subsp. piscicida, or following injection of its exotoxin. The in vivo study was complemented with an ex vivo time-lapse analysis using conditions duplicating the in vivo situation. Linings of enterocytes detached from intestine mucosa dissociate into isolated enterocytes which undergo caspase 3-mediated anoikis with cell rounding, loss of polarization, condensation of chromatin and fragmentation of the nuclear envelope, early swelling of mitochondria with rupture of the outer membrane, and brush border disappearance. One mechanism for brush border loss was shedding of apoptotic bodies incorporating the apical part of the enterocyte. Brush border disappearance was also associated with disassembly of the F-actin microvillar core and involved re-absorption into the cell, or expansion and vesiculation followed by shedding of microvillar fragments. The enterocyte anoikis terminates by secondary necrosis and lysis due to lack of elimination by phagocytosis of apoptosing enterocytes. The conditions prevailing in vivo in the gut lumen accelerate enterocyte secondary necrosis. Our results underscore the importance of analyzing anoikis under conditions similar to those occurring in vivo.

Bacterial modulation of phagocyte cell death is an emerging theme in pathogenesis. Here we describe the systemic destruction of macrophages and neutrophils by the Gram-negative Photobacterium damselae ssp. piscicida (Phdp) in fish pasteurellosis, a deadly systemic infection. Following experimental inoculation, Phdp spreads by bacteraemia and colonizes the organs, producing a septicaemic infection, and secretes the apoptogenic exotoxin AIP56 which is systemically disseminated. In experimental and natural pasteurellosis, destruction of macrophages and neutrophils by secondary necrosis following caspase-3-associated apoptosis was seen predominantly in the spleen, head kidney and gut lamina propria. Identical phagocyte destruction occurred after injection of rAIP56, but not of heat-inactivated rAIP56, or AIP56-negative Phdp strains, indicating that AIP56 is responsible for phagocyte destruction occurring in pasteurellosis. Active caspase-3 and active neutrophil elastase are present in the blood in advanced infection, indicating that phagocyte lysis by secondary necrosis is accompanied by release of tissue-damaging molecules. The AIP56-induced lysis of phagocytes represents a very efficient, self-amplifying etiopathogenic mechanism, because it results in two effects that operate in concert against the host, namely, evasion of the pathogen from a crucial defence mechanism through the destruction of both professional phagocytes, and release of tissue-damaging molecules. The induction by a bacterial exotoxin of in vivo systemic lysis of both professional phagocytes by secondary necrosis, now described for the first time, may represent an overlooked etiopathogenic mechanism operating in other infections of vertebrates.

Interleukin-12 (IL-12) is a heterodimeric cytokine pivotal in resistance to microbial and viral infections. In the search for immunoregulatory genes in sea bass the genes for the two IL-12 subunits p40 and p35 were cloned and sequenced. Molecular characterization of these two genes was performed at both the cDNA and genomic levels. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is similar to the human orthologue, whilst the sea bass IL-12 p35 gene structure, as reported for pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. The promoter analysis of both sea bass and pufferfish IL-12 genes showed the presence of the main cis-acting elements involved in the transcriptional regulation of human and mouse orthologues. The involvement of IL-12 in sea bass anti-bacterial immune responses was demonstrated by investigating the expression profiles of IL-1beta, IL-12 p40 and p35 in the head-kidney and spleen following intraperitoneal injection of UV-killed and live Photobacterium damselae ssp. piscicida (Phdp). Finally, the importance of nuclear factor (NF)-kappaB on UV-killed Phdp-induced IL-12 p40 and p35 gene transcription was shown by the use of pyrrolidine dithiocarbamate (PDTC).

Interleukin-10 (IL-10) is a pleiotropic cytokine generally known for its relevance in the resolution of inflammation, but that also has immunostimulatory properties. Here is described the isolation and characterization of the sea bass IL-10 (sbIL-10) cDNA and gene. The sbIL-10 gene encodes a 187 amino acid protein and comprises a five exon-four intron structure as other known IL-10 genes. Important structural residues are maintained in the sbIL-10 protein, including the four cysteines responsible for the two intra-chain disulfide bridges reported for human IL-10. The 3D structure of sbIL-10 was predicted. This first homology model of a fish IL-10 reveals a high degree of compatibility between the dimeric quaternary architectures of sbIL-10 and its mammalian counterparts. The phylogenetic analysis clusters sbIL-10 with other IL-10s, apart from IL-10-related molecules. The involvement of IL-10 in sea bass immune responses was demonstrated by investigating the expression profiles of IL-1beta and IL-10 in the head-kidney and spleen following intraperitoneal injection of UV-killed Photobacterium damselae ssp. piscicida. Furthermore, involvement of IL-10 in the resolution of inflammation is for the first time suggested in fish, due to the delayed maximal mRNA levels of sbIL-10 compared to those of the pro-inflammatory IL-1beta.

Caspase-9 is an initiator caspase in the apoptotic process whose function is to activate effector caspases that are downstream in the mitochondrial pathway of apoptosis. This work reports for the first time the complete sequencing and characterisation of caspase-9 in fish. A 1924bp cDNA of sea bass caspase-9 was obtained, consisting of 1308bp open reading frame coding for 435 amino acids, 199bp of the 5'-UTR and 417bp of the 3'-UTR including a canonical polyadenilation signal 10 nucleotides upstream the polyadenilation tail. The sequence retains the pentapeptide active-site motif (QACGG) and the putative cleavage sites at Asp(121), Asp(325) and Asp(343). The sequence of sea bass caspase-9 exhibits a very close homology to the sequences of caspase-9 from other vertebrates, particularly with the putative caspases-9 of Danio rerio and Tetraodon nigroviridis (77.5 and 75.4% similarity, respectively), justifying the fact that the phylogenetic analysis groups these species together with sea bass. The sea bass caspase-9 gene exists as a single copy gene and is organised in 9 introns and 10 exons. The sea bass caspase-9 showed a basal expression in all the organs analysed, although weaker in spleen. The expression of sea bass caspase-9 in the head kidney of sea bass infected with the Photobacterium damselae ssp. piscicida (Phdp) strain PP3, showed increased expression from 0 to 12h returning to control levels at 24h. Caspase-9 activity was detected in Phdp infected sea bass head kidney from 18 to 48h post-infection, when the fish were with advanced septicaemia.

Caspase-3 is one of the major caspases operating in apoptosis, cleaving and inactivating a number of molecules and largely contributing to the apoptotic phenotype and the dismantling of the apoptoting cell. The opening reading frame of sea bass (Dicentrarchus labrax L.) caspase-3 has 281 amino acids. The complete sequence of caspase-3 shows a very close homology to the correspondent sequence from other vertebrates, in particularly with that of Takifugu rubripes and Oryzias latipes, with 87.7 and 87.9% of similarity, respectively. Furthermore, the sea bass caspase-3 sequence retains the motifs that are functionally important, such as the pentapeptide active-site motif (QACRG) and the putative cleavage sites at the aspartic acids. In the sea bass genome, the caspase-3 gene exists as a single copy gene and is organised in six exons and five introns. A very low expression of caspase-3 was detected by RT-PCR in various organs of non-stimulated sea bass, with slightly higher levels in thymus and heart and was increased in head kidneys of Photobacterium damselae ssp. piscicida infected sea bass. This increased expression was accompanied by the occurrence of high numbers of apoptoting cells with activated caspase-3.

Background: Dermatophagoides pteronyssinus, Dermatophagoides farinae and Blomia tropicalis dust mites are among the most important agents of hypersensitivity reactions in human beings. However, a role of other mites in the etiology of these reactions has not yet been excluded., Objectives: To investigate the nature of the dust mite fauna and the presence of Der p 1 (allergen 1 of Dermatophagoides pteronyssinus) and Blo t 5 (allergen 5 of Blomia tropicalis) on beds used by individuals with different socioeconomic backgrounds in Salvador, a major Brazilian city and to investigate possible associations of mite frequencies and allergen levels with (a) season of the year, (b) housing characteristics, (c) bed cleaning behaviors that could affect mite densities, and (d) allergy history., Methods: Dust samples were collected from 459 beds of 101 residences from two groups with different socioeconomic levels (hereafter called wealthy and poor groups) in the city of Salvador, Brazil, for the identification of mite species and determination of Der p 1 and Blo t 5 levels. History of allergy was collected using the ISAAC phase I questionnaire., Results: Eighty nine percent of the beds analyzed harbored at least one mite species. B. tropicalis was found in 71.8%, D. pteronyssinus in 39.9%, Cheyletus sp. in 33.9%, and Gohieria fusca in 21.1% of the beds. B. tropicalis was found with a similar frequency in beds of the two socioeconomic groups; D. pteronyssinus was found more frequently in the beds of the wealthy than of the poor group, whereas the reverse was observed with G. fusca. The concentrations of Der p 1 and Blo t 5 allergens exceeded the cut-off for sensitization of 2 microg/g of dust in 94.0% and 69.3% of the wealthy and poor group beds, respectively. No associations were found between history of allergy and mite species or between history of allergy and the concentrations of mite allergens., Conclusions: The observation of B. tropicalis and D. pteronyssinus as the most frequently found mites is consistent with previous reports from tropical regions. The higher frequency of G. fusca in beds of individuals from the poor group than those from the wealthy group could be a consequence of different bed cleaning behaviors between the two groups.

A strategy used by extracellular pathogens to evade phagocytosis is the utilization of exotoxins that kill host phagocytes. We have recently shown that one major pathogenicity strategy of Photobacterium damselae subsp. piscicida (Phdp), the agent of the widespread fish pasteurellosis, is the induction of extensive apoptosis of sea bass macrophages and neutrophils that results in lysis of these phagocytes by post-apoptotic secondary necrosis. Here we show that this unique process is mediated by a novel plasmid-encoded apoptosis inducing protein of 56 kDa (AIP56), an exotoxin abundantly secreted by all virulent, but not avirulent, Phdp strains tested. AIP56 is related to an unknown protein of the enterohemorrhagic Escherichia coli O157:H7 and NleC, a Citrobacter rodentium type III secreted effector of unknown function. Passive immunization of sea bass with a rabbit anti-AIP56 serum conferred protection against Phdp challenge, indicating that AIP56 represents a key virulence factor of that pathogen and is a candidate for the design of an anti-pasteurellosis vaccine.

Photobacterium damselae subsp. piscicida is the causative agent of pasteurellosis in wild and farmed marine fish worldwide. Although serologically homogeneous, recent molecular advances have led to the discovery of distinct genetic clades, depending on geographical origin. Further details of the strategies for host colonisation have arisen including information on the role of capsule, susceptibility to oxidative stress, confirmation of intracellular survival in host epithelial cells, and induced apoptosis of host macrophages. This improved understanding has given rise to new ideas and advances in vaccine technologies, which are reviewed in this paper.

Mycobacteriosis (piscine tuberculosis) has been reported to affect a wide range of freshwater and marine fish species; however, this is the first report describing mycobacterial infections in turbot Scophthalmus maximus. High numbers of granulomas were initially observed in the organs of moribund farmed turbot. Bacteriological analysis of organs with granulomas led to the isolation of Mycobacterium marinum. Further analysis, to determine the prevalence of the infection in the farm and to identify its source, showed the occurrence of a dual infection by M. marinum and M. chelonae. The presence of Nocardia sp. in some of the fish infected with mycobacteria was also detected. The presence of granulomas in internal organs of apparently healthy fish indicated a high prevalence of the disease, a conclusion that was supported by isolating mycobacteria from all fish with or without granulomas. The infection was probably responsible for the mortality observed (approximately 2% mo(-1)), as most of the recently dead fish presented high numbers of granulomas and isolation of mycobacteria was possible from all of the fish. The isolation of M. marinum from the inlet water suggested this as the most plausible source for the infection occurring in the farm.