[the Use Of Fish On Buccal Smear To Investigate Mosaicism With A 45,x Cell Line: Study On Healthy Men And Patients With Disorders Of Sex Development].

To verify whether fluorescence in situ hybridization (FISH) of cells from the buccal epithelium could be employed to detect cryptomosaicism with a 45,X lineage in 46,XY patients. Samples of nineteen 46,XY healthy young men and five patients with disorders of sex development (DSD), four 45,X/46,XY and one 46,XY were used. FISH analysis with X and Y specific probes on interphase nuclei from blood lymphocytes and buccal epithelium were analyzed to investigate the proportion of nuclei containing only the signal of the X chromosome. The frequency of nuclei containing only the X signal in the two tissues of healthy men did not differ (p = 0.69). In all patients with DSD this frequency was significantly higher, and there was no difference between the two tissues (p = 0.38), either. Investigation of mosaicism with a 45,X cell line in patients with 46,XY DSD or sterility can be done by FISH directly using cells from the buccal epithelium.

Feasibility And Reproducibility Of Diffusion-weighted Magnetic Resonance Imaging Of The Fetal Brain In Twin-twin Transfusion Syndrome.

The aim of this study is to test the feasibility and reproducibility of diffusion-weighted magnetic resonance imaging (DW-MRI) evaluations of the fetal brains in cases of twin-twin transfusion syndrome (TTTS). From May 2011 to June 2012, 24 patients with severe TTTS underwent MRI scans for evaluation of the fetal brains. Datasets were analyzed offline on axial DW images and apparent diffusion coefficient (ADC) maps by two radiologists. The subjective evaluation was described as the absence or presence of water diffusion restriction. The objective evaluation was performed by the placement of 20-mm(2) circular regions of interest on the DW image and ADC maps. Subjective interobserver agreement was assessed by the kappa correlation coefficient. Objective intraobserver and interobserver agreements were assessed by proportionate Bland-Altman tests. Seventy-four DW-MRI scans were performed. Sixty of them (81.1%) were considered to be of good quality. Agreement between the radiologists was 100% for the absence or presence of diffusion restriction of water. For both intraobserver and interobserver agreement of ADC measurements, proportionate Bland-Altman tests showed average percentage differences of less than 1.5% and 95% CI of less than 18% for all sites evaluated. Our data demonstrate that DW-MRI evaluation of the fetal brain in TTTS is feasible and reproducible.

Evidences Of Endocytosis Via Caveolae Following Blood-brain Barrier Breakdown By Phoneutria Nigriventer Spider Venom.

Spider venoms contain neurotoxic peptides aimed at paralyzing prey or for defense against predators; that is why they represent valuable tools for studies in neuroscience field. The present study aimed at identifying the process of internalization that occurs during the increased trafficking of vesicles caused by Phoneutria nigriventer spider venom (PNV)-induced blood-brain barrier (BBB) breakdown. Herein, we found that caveolin-1α is up-regulated in the cerebellar capillaries and Purkinje neurons of PNV-administered P14 (neonate) and 8- to 10-week-old (adult) rats. The white matter and granular layers were regions where caveolin-1α showed major upregulation. The variable age played a role in this effect. Caveolin-1 is the central protein that controls caveolae formation. Caveolar-specialized cholesterol- and sphingolipid-rich membrane sub-domains are involved in endocytosis, transcytosis, mechano-sensing, synapse formation and stabilization, signal transduction, intercellular communication, apoptosis, and various signaling events, including those related to calcium handling. PNV is extremely rich in neurotoxic peptides that affect glutamate handling and interferes with ion channels physiology. We suggest that the PNV-induced BBB opening is associated with a high expression of caveolae frame-forming caveolin-1α, and therefore in the process of internalization and enhanced transcytosis. Caveolin-1α up-regulation in Purkinje neurons could be related to a way of neurons to preserve, restore, and enhance function following PNV-induced excitotoxicity. The findings disclose interesting perspectives for further molecular studies of the interaction between PNV and caveolar specialized membrane domains. It proves PNV to be excellent tool for studies of transcytosis, the most common form of BBB-enhanced permeability.

Bile And Liver Metallothionein Behavior In Copper-exposed Fish.

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2mgL(-1)) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC-ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.

Triggering Of Protection Mechanism Against Phoneutria Nigriventer Spider Venom In The Brain.

Severe accidents caused by the armed spider Phoneutria nigriventer cause neurotoxic manifestations in victims. In experiments with rats, P. nigriventer venom (PNV) temporarily disrupts the properties of the BBB by affecting both the transcellular and the paracellular route. However, it is unclear how cells and/or proteins participate in the transient opening of the BBB. The present study demonstrates that PNV is a substrate for the multidrug resistance protein-1 (MRP1) in cultured astrocyte and endothelial cells (HUVEC) and increases mrp1 and cx43 and down-regulates glut1 mRNA transcripts in cultured astrocytes. The inhibition of nNOS by 7-nitroindazole suggests that NO derived from nNOS mediates some of these effects by either accentuating or opposing the effects of PNV. In vivo, MRP1, GLUT1 and Cx43 protein expression is increased differentially in the hippocampus and cerebellum, indicating region-related modulation of effects. PNV contains a plethora of Ca(2+), K(+) and Na(+) channel-acting neurotoxins that interfere with glutamate handling. It is suggested that the findings of the present study are the result of a complex interaction of signaling pathways, one of which is the NO, which regulates BBB-associated proteins in response to PNV interference on ions physiology. The present study provides additional insight into PNV-induced BBB dysfunction and shows that a protective mechanism is activated against the venom. The data shows that PNV has qualities for potential use in drug permeability studies across the BBB.

Behavioral Changes Caused By Austrodiplostomum Spp. In Hoplias Malabaricus From The São Francisco River, Brazil.

Traira (Hoplias malabaricus) is a neotropical fish that is widely distributed in freshwater environments in South America. In the present study, we documented the occurrence of metacercariae of Austrodiplostomum spp. (Diplostomidae) in the eyes and cranial cavity of H. malabaricus and described parasite-induced behavioral changes in the host. The fish were collected from the upper São Francisco River, in the Serra da Canastra mountain range, Minas Gerais, transported alive to the laboratory, observed for 2 weeks, and subsequently examined for parasites. Of the 35 fish examined, 28 (80 %) had free metacercariae in the vitreous humor (mean intensity=95.4; mean abundance=76.3), and 24 (68.57 %) had free metacercariae in the cranial cavity, mainly concentrated below the floor of the brain, at the height of the ophthalmic lobe (mean intensity=12.91; mean abundance=8.85). Specimens of H. malabaricus with a high intensity of infection in the brain displayed changes in swimming behavior.

Vascular Endothelial Growth Factor Increases During Blood-brain Barrier-enhanced Permeability Caused By Phoneutria Nigriventer Spider Venom.

Phoneutria nigriventer spider accidental envenomation provokes neurotoxic manifestations, which when critical, results in epileptic-like episodes. In rats, P. nigriventer venom (PNV) causes blood-brain barrier breakdown (BBBb). The PNV-induced excitotoxicity results from disturbances on Na(+), K(+) and Ca(2+) channels and glutamate handling. The vascular endothelial growth factor (VEGF), beyond its angiogenic effect, also, interferes on synaptic physiology by affecting the same ion channels and protects neurons from excitotoxicity. However, it is unknown whether VEGF expression is altered following PNV envenomation. We found that adult and neonates rats injected with PNV showed immediate neurotoxic manifestations which paralleled with endothelial occludin, β-catenin, and laminin downregulation indicative of BBBb. In neonate rats, VEGF, VEGF mRNA, and Flt-1 receptors, glutamate decarboxylase, and calbindin-D28k increased in Purkinje neurons, while, in adult rats, the BBBb paralleled with VEGF mRNA, Flk-1, and calbindin-D28k increases and Flt-1 decreases. Statistically, the variable age had a role in such differences, which might be due to age-related unequal maturation of blood-brain barrier (BBB) and thus differential cross-signaling among components of the glial neurovascular unit. The concurrent increases in the VEGF/Flt-1/Flk-1 system in the cerebellar neuron cells and the BBBb following PNV exposure might imply a cytokine modulation of neuronal excitability consequent to homeostatic perturbations induced by ion channels-acting PNV neuropeptides. Whether such modulation represents neuroprotection needs further investigation.

Not Simply A Matter Of Fish Intake.

Background and aims: Recent findings have highlighted enhanced fish consumption as a potential measure to increase intake of healthy fatty acids, particularly omega-3. The generalizability of this recommendation, however, may fall short by differences in fish species and cooking techniques. Hence, we investigated how these 2 variables affect the lipid content in fish flesh. Methods and Results: Nine species of freshwater, deep sea or shore fish were grilled, steamed or fried with or without the addition of soybean oil, olive oil or butter. The lipid composition was analysed and a significant difference was observed in cholesterol, saturated fatty acids, polyunsaturated fatty acids, omega-3 fatty acids, and omega-6 fatty acids contents between species (p<0.05). The use of soybean or olive oil was associated with a significant change in flesh concentration of polyunsaturated, omega-3 and omega-6 fatty acids (p<0.05). Conclusion: This study calls attention to the specific lipid content that must be expected from different fish species and cooking techniques.

Effects Of Partial Inhibition Of Respiratory Complex I On H2o 2 Production By Isolated Brain Mitochondria In Different Respiratory States.

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.

Intrahippocampal Infusion Of Crotamine Isolated From Crotalus Durissus Terrificus Alters Plasma And Brain Biochemical Parameters.

Crotamine is one of the main constituents of the venom of the South American rattlesnake Crotalus durissus terrificus. Here we sought to investigate the inflammatory and toxicological effects induced by the intrahippocampal administration of crotamine isolated from Crotalus whole venom. Adult rats received an intrahippocampal infusion of crotamine or vehicle and were euthanized 24 h or 21 days after infusion. Plasma and brain tissue were collected for biochemical analysis. Complete blood count, creatinine, urea, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), creatine-kinase (CK), creatine kinase-muscle B (CK-MB) and oxidative parameters (assessed by DNA damage and micronucleus frequency in leukocytes, lipid peroxidation and protein carbonyls in plasma and brain) were quantified. Unpaired and paired t-tests were used for comparisons between saline and crotamine groups, and within groups (24 h vs. 21 days), respectively. After 24 h crotamine infusion promoted an increase of urea, GOT, GPT, CK, and platelets values (p ≤ 0.01), while red blood cells, hematocrit and leukocytes values decreased (p ≤ 0.01). Additionally, 21 days after infusion crotamine group showed increased creatinine, leukocytes, TBARS (plasma and brain), carbonyl (plasma and brain) and micronucleus compared to the saline-group (p ≤ 0.01). Our findings show that crotamine infusion alter hematological parameters and cardiac markers, as well as oxidative parameters, not only in the brain, but also in the blood, indicating a systemic pro-inflammatory and toxicological activity. A further scientific attempt in terms of preserving the beneficial activity over toxicity is required.

Rotenone Exerts Similar Stimulatory Effects On H2o2 Production By Isolated Brain Mitochondria From Young-adult And Old Rats.

Chronic and systemic treatment of rodents with rotenone, a classical inhibitor of mitochondrial respiratory complex I, results in neurochemical, behavioral, and neuropathological features of Parkinson's disease. The aim of the present study was to evaluate whether brain mitochondria from old rats (24 months old) would be more susceptible to rotenone-induced inhibition of oxygen consumption and increased generation of H2O2 than mitochondria from young-adult rats (3-4 months old). Isolated brain mitochondria were incubated in the presence of different rotenone concentrations (5, 10, and 100nM), and oxygen consumption and H2O2 production were measured during respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration). Respiratory state 3 and citrate synthase activity were significantly lower in mitochondria from old rats. Mitochondria from young-adult and old rats showed similar sensitivity to rotenone-induced inhibition of oxygen consumption. Similarly, H2O2 production rates by both types of mitochondria were dose-dependently stimulated to the same extent by increasing concentrations of rotenone. We conclude that rotenone exerts similar effects on oxygen consumption and H2O2 production by isolated brain mitochondria from young-adult and old rats. Therefore, aging does not increase the mitochondrial H2O2 generation in response to complex I inhibition.

Fish Biodiversity Of The Vitória-trindade Seamount Chain, Southwestern Atlantic: An Updated Database.

Despite a strong increase in research on seamounts and oceanic islands ecology and biogeography, many basic aspects of their biodiversity are still unknown. In the southwestern Atlantic, the Vitória-Trindade Seamount Chain (VTC) extends ca. 1,200 km offshore the Brazilian continental shelf, from the Vitória seamount to the oceanic islands of Trindade and Martin Vaz. For a long time, most of the biological information available regarded its islands. Our study presents and analyzes an extensive database on the VTC fish biodiversity, built on data compiled from literature and recent scientific expeditions that assessed both shallow to mesophotic environments. A total of 273 species were recorded, 211 of which occur on seamounts and 173 at the islands. New records for seamounts or islands include 191 reef fish species and 64 depth range extensions. The structure of fish assemblages was similar between islands and seamounts, not differing in species geographic distribution, trophic composition, or spawning strategies. Main differences were related to endemism, higher at the islands, and to the number of endangered species, higher at the seamounts. Since unregulated fishing activities are common in the region, and mining activities are expected to drastically increase in the near future (carbonates on seamount summits and metals on slopes), this unique biodiversity needs urgent attention and management.

Ecotoxicological Effects Of Carbofuran And Oxidised Multiwalled Carbon Nanotubes On The Freshwater Fish Nile Tilapia: Nanotubes Enhance Pesticide Ecotoxicity.

The interactions of carbon nanotubes with pesticides, such as carbofuran, classical contaminants (e.g., pesticides, polyaromatic hydrocarbons, heavy metals, and dyes) and emerging contaminants, including endocrine disruptors, are critical components of the environmental risks of this important class of carbon-based nanomaterials. In this work, we studied the modulation of acute carbofuran toxicity to the freshwater fish Nile tilapia (Oreochromis niloticus) by nitric acid treated multiwalled carbon nanotubes, termed HNO3-MWCNT. Nitric acid oxidation is a common chemical method employed for the purification, functionalisation and aqueous dispersion of carbon nanotubes. HNO3-MWCNT were not toxic to Nile tilapia at concentrations ranging from 0.1 to 3.0 mg/L for exposure times of up to 96 h. After 24, 48, 72 and 96 h, the LC50 values of carbofuran were 4.0, 3.2, 3.0 and 2.4 mg/mL, respectively. To evaluate the influence of carbofuran-nanotube interactions on ecotoxicity, we exposed the Nile tilapia to different concentrations of carbofuran mixed together with a non-toxic concentration of HNO3-MWCNT (1.0 mg/L). After 24, 48, 72, and 96 h of exposure, the LC50 values of carbofuran plus nanotubes were 3.7, 1.6, 0.7 and 0.5 mg/L, respectively. These results demonstrate that HNO3-MWCNT potentiate the acute toxicity of carbofuran, leading to a more than five-fold increase in the LC50 values. Furthermore, the exposure of Nile tilapia to carbofuran plus nanotubes led to decreases in both oxygen consumption and swimming capacity compared to the control. These findings indicate that carbon nanotubes could act as pesticide carriers affecting fish survival, metabolism and behaviour.

Application Of Laser Microdissection Icp-ms For High Resolution Elemental Mapping In Mouse Brain Tissue: A Comparative Study With Laser Ablation Icp-ms.

Mapping of elements in biological tissue by laser induced mass spectrometry is a fast growing analytical methodology in life sciences. This method provides a multitude of useful information of metal, nonmetal, metalloid and isotopic distribution at major, minor and trace concentration ranges, usually with a lateral resolution of 12-160 µm. Selected applications in medical research require an improved lateral resolution of laser induced mass spectrometric technique at the low micrometre scale and below. The present work demonstrates the applicability of a recently developed analytical methodology - laser microdissection associated to inductively coupled plasma mass spectrometry (LMD ICP-MS) - to obtain elemental images of different solid biological samples at high lateral resolution. LMD ICP-MS images of mouse brain tissue samples stained with uranium and native are shown, and a direct comparison of LMD and laser ablation (LA) ICP-MS imaging methodologies, in terms of elemental quantification, is performed.