Immunity and inflammation in status epilepticus and its sequelae: possibilities for therapeutic application.

Status epilepticus (SE) is a life-threatening neurological emergency often refractory to available treatment options. It is a very heterogeneous condition in terms of clinical presentation and causes, which besides genetic, vascular and other structural causes also include CNS or severe systemic infections, sudden withdrawal from benzodiazepines or anticonvulsants and rare autoimmune etiologies. Treatment of SE is essentially based on expert opinions and antiepileptic drug treatment per se seems to have no major impact on prognosis. There is, therefore, urgent need of novel therapies that rely upon a better understanding of the basic mechanisms underlying this clinical condition. Accumulating evidence in animal models highlights that inflammation ensuing in the brain during SE may play a determinant role in ongoing seizures and their long-term detrimental consequences, independent of an infection or auto-immune cause; this evidence encourages reconsideration of the treatment flow in SE patients.

Theory of mind impairment in patients with behavioural variant fronto-temporal dementia (bv-FTD) increases caregiver burden.

BACKGROUND: Theory of mind (ToM), the capacity to infer the intention, beliefs and emotional states of others, is frequently impaired in behavioural variant fronto-temporal dementia patients (bv-FTDp); however, its impact on caregiver burden is unexplored. SETTING: National Institute of Neurological Disorders and Stroke, National Institutes of Health. SUBJECTS: bv-FTDp (n = 28), a subgroup of their caregivers (n = 20) and healthy controls (n = 32). METHODS: we applied a faux-pas (FP) task as a ToM measure in bv-FTDp and healthy controls and the Zarit Burden Interview as a measure of burden in patients' caregivers. Patients underwent structural MRI; we used voxel-based morphometry to examine relationships between regional atrophy and ToM impairment and caregiver burden. RESULTS: FP task performance was impaired in bv-FTDp and negatively associated with caregiver burden. Atrophy was found in areas involved in ToM. Caregiver burden increased with greater atrophy in left lateral premotor cortex, a region associated in animal models with the presence of mirror neurons, possibly involved in empathy. CONCLUSION: ToM impairment in bv-FTDp is associated with increased caregiver burden.

Lipid mobilization and gluconeogenesis in plants: Do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

Furosemide stimulation of parathormone in humans: role of the calcium-sensing receptor and the renin-angiotensin system.

Interactions between sodium and calcium regulating systems are poorly characterized but clinically important. Parathyroid hormone (PTH) levels are increased shortly after furosemide treatment by an unknown mechanism, and this effect is blunted by the previous administration of a calcimimetic in animal studies. Here, we explored further the possible underlying mechanisms of this observation in a randomized crossover placebo-controlled study performed in 18 human males. Volunteers took either cinacalcet (60 mg) or placebo and received a 20 mg furosemide injection 3 h later. Plasma samples were collected at 15-min intervals and analyzed for intact PTH, calcium, sodium, potassium, magnesium, phosphate, plasma renin activity (PRA), and aldosterone up to 6 h after furosemide injection. Urinary electrolyte excretion was also monitored. Subjects under placebo presented a sharp increase in PTH levels after furosemide injection. In the presence of cinacalcet, PTH levels were suppressed and marginal increase of PTH was observed. No significant changes in electrolytes and urinary excretion were identified that could explain the furosemide-induced increase in PTH levels. PRA and aldosterone were stimulated by furosemide injection but were not affected by previous cinacalcet ingestion. Expression of NKCC1, but not NKCC2, was found in parathyroid tissue. In conclusion, our results indicate that furosemide acutely stimulates PTH secretion in the absence of any detectable electrolyte changes in healthy adults. A possible direct effect of furosemide on parathyroid gland needs further studies.

Macroporous Scaffolds for Bone Engineering. Studies on Cell Culture and Ectopic Bone Formation

Bone engineering is a rapidly developing area of reconstructive medicine where bone inducing factors and/or cells are combined with a scaffold material to regenerate the structure and function of the original tissue. The aim of this study was to compare the suitability of different macroporous scaffold types for bone engineering applications. The two scaffold categories studied were a) the mechanically strong and stable titanium fiber meshes and b) the elastic and biodegradable porous polymers. Furthermore, bioactive modifications were applied to these basic scaffold types, and their effect on the osteogenic responses was evaluated in cell culture and ectopic bone formation studies. The osteogenic phenotype of cultured cell-scaffold constructs was heightened with a sol-gel derived titania coating, but not with a mixed titania-silica coating. The latter coating also resulted in delayed ectopic bone formation in bone marrow stromal cell seeded scaffolds. However, the better bone contact in early implantation times and more even bone tissue distribution at later times indicated enhanced osteoconductivity of both the coated scaffold types. Overall, the most promising bone engineering results were obtained with titania coated fiber meshes. Elastic and biodegradable poly(ε-caprolactone/D,L-lactide) based scaffolds were also developed in this study. The degradation rates of the scaffolds in vitro were governed by the hydrophilicity of the polymer matrix, and the porous architecture was controlled by the amount and type of porogen used. A continuous phase macroporosity was obtained using a novel CaCl2 • 6H2O porogen. Dynamic culture conditions increased cell invasion, but decreased cell numbers and osteogenicity, within the scaffolds. Osteogenic differentiation in static cultures and ectopic bone formation in cell seeded scaffolds were enhanced in composites, with 30 wt-% of bioactive glass filler.

Galanin pathogenic mutations in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is a common epilepsy syndrome with a complex etiology. Despite evidence for the participation of genetic factors, the genetic basis of TLE remains largely unknown. A role for the galanin neuropeptide in the regulation of epileptic seizures has been established in animal models more than two decades ago. However, until now there was no report of pathogenic mutations in GAL, the galanin-encoding gene, and therefore its role in human epilepsy was not established. Here, we studied a family with a pair of monozygotic twins affected by TLE and two unaffected siblings born to healthy parents. Exome sequencing revealed that both twins carried a novel de novo mutation (p.A39E) in the GAL gene. Functional analysis revealed that the p.A39E mutant showed antagonistic activity against galanin receptor 1 (GalR1)-mediated response, and decreased binding affinity and reduced agonist properties for GalR2. These findings suggest that the p.A39E mutant could impair galanin signaling in the hippocampus, leading to increased glutamatergic excitation and ultimately to TLE. In a cohort of 582 cases, we did not observe any pathogenic mutations indicating that mutations in GAL are a rare cause of TLE. The identification of a novel de novo mutation in a biologically-relevant candidate gene, coupled with functional evidence that the mutant protein disrupts galanin signaling, strongly supports GAL as the causal gene for the TLE in this family. Given the availability of galanin agonists which inhibit seizures, our findings could potentially have direct implications for the development of anti-epileptic treatment.

Long-term treatment with insulin induces apoptosis in brown adipocytes: role of oxidative stress

Trying to define the precise role played by insulin regulating the survival of brown adipocytes, we have used rat fetal brown adipocytes maintained in primary culture. The effect of insulin on apoptosis and the mechanisms involved were assessed. Different from the known effects of insulin as a survival factor, we have found that long-term treatment (72 h) with insulin induces apoptosis in rat fetal brown adipocytes. This process is dependent on the phosphatidylinositol 3-kinase/mammalian target of rapamycin/p70 S6 kinase pathway. Short-term treatment with the conditioned medium from brown adipocytes treated with insulin for 72 h mimicked the apoptotic effect of insulin. During the process, caspase 8 activation, Bid cleavage, cytochrome c release, and activation of caspases 9 and 3 are sequentially produced. Treatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Z-VAD), prevents activation of this apoptotic cascade. The antioxidants, ascorbic acid and superoxide dismutase, also impair this process of apoptosis. Moreover, generation of reactive oxygen species (ROS), probably through reduced nicotinamide adenine dinucleotide phosphate oxidases, and a late decrease in reduced glutathione content are produced. According to this, antioxidants prevent caspase 8 activation and Bid cleavage, suggesting that ROS production is an important event mediating this process of apoptosis. However, the participation of uncoupling protein-1, -2, and -3 regulating ROS is unclear because their levels remain unchanged upon insulin treatment for 72 h. Our data suggest that the prolonged hyperinsulinemia might cause insulin resistance through the loss of brown adipose tissue.

Use of a ventricular septal defect occluder for apical closure in transapical aortic valve replacement.

During transapical transcatheter aortic valve replacement (TA-TAVR), the apical closure remains a challenge for the surgeon, having the risk for ventricular tear and massive bleeding. Apical closure devices are already under clinical evaluation, but only a few can lead to a full percutaneous TA-TAVR. We describe the successful use of a 9-mm myocardial occluder (ventricular septal defect occluder) that was used to seal the apex after a standard TA-TAVR (using the Sapien XT 23-mm transcatheter valve and the Ascendra + delivery system). The placement of the nonmodified myocardial occluder was performed through the Ascendra + delivery system, with a very small amount of blood loss and an acceptable sealing of the apical tear. This approach is feasible and represents a further step toward true-percutaneous transapical heart valve procedures. Modified apical occluders are under evaluation in animal models.

Dose calibrator linearity test: 99mTc versus 18F radioisotopes

Objective: The present study was aimed at evaluating the viability of replacing 18F with 99mTc in dose calibrator linearity testing. Materials and Methods: The test was performed with sources of 99mTc (62 GBq) and 18F (12 GBq) whose activities were measured up to values lower than 1 MBq. Ratios and deviations between experimental and theoretical 99mTc and 18F sources activities were calculated and subsequently compared. Results: Mean deviations between experimental and theoretical 99mTc and 18F sources activities were 0.56 (± 1.79)% and 0.92 (± 1.19)%, respectively. The mean ratio between activities indicated by the device for the 99mTc source as measured with the equipment pre-calibrated to measure 99mTc and 18F was 3.42 (± 0.06), and for the 18F source this ratio was 3.39 (± 0.05), values considered constant over the measurement time. Conclusion: The results of the linearity test using 99mTc were compatible with those obtained with the 18F source, indicating the viability of utilizing both radioisotopes in dose calibrator linearity testing. Such information in association with the high potential of radiation exposure and costs involved in 18F acquisition suggest 99mTc as the element of choice to perform dose calibrator linearity tests in centers that use 18F, without any detriment to the procedure as well as to the quality of the nuclear medicine service.

Mechanisms of protein homeostasis in health, aging and disease.

When emerging from the ribosomes, new polypeptides need to fold properly, eventually translocate, and then assemble into stable, yet functionally flexible complexes. During their lifetime, native proteins are often exposed to stresses that can partially unfold and convert them into stably misfolded and aggregated species, which can in turn cause cellular damage and propagate to other cells. In animal cells, especially in aged neurons, toxic aggregates may accumulate, induce cell death and lead to tissue degeneration via different mechanisms, such as apoptosis as in Parkinson's and Alzheimer's diseases and aging in general. The main cellular mechanisms effectively controlling protein homeostasis in youth and healthy adulthood are: (1) the molecular chaperones, acting as aggregate unfolding and refolding enzymes, (2) the chaperone-gated proteases, acting as aggregate unfolding and degrading enzymes, (3) the aggresomes, acting as aggregate compacting machineries, and (4) the autophagosomes, acting as aggregate degrading organelles. For unclear reasons, these cellular defences become gradually incapacitated with age, leading to the onset of degenerative diseases. Understanding these mechanisms and the reasons for their incapacitation in late adulthood is key to the design of new therapies against the progression of aging, degenerative diseases and cancers.

Sistema em fluxo para determinação espectrofotométrica de uréia em plasma de sangue animal empregando leguminosa como fonte natural da enzima urease

A spectrophotometric flow injection analysis (FIA) procedure employing natural urease enzyme source for the determination of urea in animal blood plasma was developed. Among leguminous plants used in the Brazilian agriculture, the Cajanus cajan specie was selected as urease source considering its efficiency and availability. A minicolumn was filled with leguminous fragments and coupled to the FIA manifold, where urea was on-line converted to ammonium ions and subsequently it was quantified by spectrophotometry. The system was employed to determine urea in animal plasma samples without any prior treatment. Accuracy was assessed by comparison results with those obtained employing the official procedure and no significant difference at 90 % confidence level was observed. Other profitable features such as an analytical throughput of 30 determinations per hour, a reagent consumption of 19.2 mg sodium salicylate, 0.5 mg sodium hipochloride and a relative standard deviation of 1.4 % (n= 12) were also obtained.

Behavioral Responses in Mice Selectively Bred for High and Low Levels of Open-Field Thigmotaxis

In animal psychology, the open-field (OF) test is a traditional method for studying different aspects of rodent behavior, with thigmotaxis (i.e., wallseeking behavior) being one of the best validated OF parameters employed to measure emotionality. The main purpose of the present study was to investigate the selection response in mice selectively bred for high and low levels of OF thigmotaxis (the HOFT and LOFT lines, respectively). The mice (N = 2048) were selected for 23 generations, resulting in bidirectional phenotypic divergence between the two lines; that is, the HOFT mice were more thigmotactic (i.e., more emotional) than the LOFT mice across the different generations. The origin of the line difference in thigmotaxis was further investigated by using the crossfostering paradigm, with the results suggesting that the divergence between the two lines was primarily innate in origin and not influenced by differing maternal behavior. The stability of the selection trait was examined by testing the animals at different ages as well as in varying conditions. The results indicated that the line difference in thigmotaxis was not affected by age at the time of testing, and it also persisted in the different OF testing situations as well as during pregnancy and lactation. The examination of a possible coselection of other characteristics revealed that the more thigmotactic HOFT mice lived longer than the less thigmotactic LOFT mice. In addition, the HOFT mice tended to rear and explore less than the LOFT mice, supporting the general assumption that emotionality and exploration are inversely related. The two lines did not generally differ in ambulation and defecation, that is, in the traditional OF indexes of emotionality, conforming to the suggestion that emotionality is a multidimensional construct. The effects of sex on different OF parameters were also assessed, with the results suggesting that among the HOFT and LOFT lines, the female mice were more emotional than the male mice. The examination of the temporal changes in the HOFT and LOFT lines’ OF behavior revealed some contradictory findings that also partially conflicted with general assumptions. Although this study did not show prominent differences in maternal responsiveness between the HOFT and LOFT mothers, the results suggested that the line divergence in emotionality was more pronounced in the presence of a pup after parturition than during pregnancy. The present study clearly demonstrates that OF thigmotaxis is a strong characteristic for producing two diverging lines of mice. The difference in thigmotaxis between the selectively bred HOFT and LOFT mice seemed to be a stable and robust feature of these animals, and it appeared to stem from a genetic background.

Metabolic response induced by endophytic fungi and bacteria in H. marrubioides Epling in vitro microplants

Hyptis marrubioides Epling is a native plant from Brazilian Cerrado. In this paper, the response of in vitro microplants of this species to inoculation with bacterial and fungal endophytic isolates is evaluated. HPLC-DAD analysis showed the presence of 3,4-O-(Z)-dicaffeoylquinic acid and quercetin-7-O-glucoside as the main components. GC/MS analysis demonstrated that the sesquiterpenes τ-cadinol and caryophyllene oxide were only produced in microplants inoculated with endophytic bacteria, while methyl hexadecanoate, methyl heptadecanoate and methyl (Z,Z,Z) 9,12,15-octadecatrienoate and the triterpene methyl 3β-hydroxy-urs-12-en-28-oate were overexpressed only when the microplant was treated with endophytic fungi.

Biomolecules produced in liquid-state fermentation by a marine-derived fungus, Penicillium roqueforti

Screening of biomass of a new marine-derived strain of Penicillium roqueforti, as produced by liquid-state fermentation, led to the identification of several volatile organic compounds active in the fatty acid pathway as well as fragments produced by their catabolism, terpenoids, and metabolites from the shikimic acid pathway. In addition, five non-volatile organic compounds, triolein, ergosterol peroxide, 9(11)-dehydroergosterol peroxide, 4-hydroxybenzaldehyde, and d-mannitol, were isolated and identified by spectroscopy. The results showed that this fungal strain did not produce any mycotoxin in the culture conditions applied, and thus is useful for industrial applications, where high value-added biomolecules are generated.