Modulating Expression of Endogenous Interleukin 1 Beta in the Acute Phase of the Pilocarpine Model of Epilepsy May Change Animal Survival.

The pilocarpine-induced (PILO) model has helped elucidate the electrophysiological and molecular aspects related to mesial temporal lobe epilepsy. It has been suggested that the extensive cell death and edema observed in the brains of these animals could be induced by increased inflammatory responses, such as the rapid release of the inflammatory cytokine interleukin 1 beta (Il1b). In this study, we investigate the role of endogenous Il1b in the acute phase of the PILO model. Our aim is twofold. First, we want to determine whether it is feasible to silence Il1b in the central nervous system using a non-invasive procedure. Second, we aim to investigate the effect of silencing endogenous Il1b and its antagonist, Il1rn.We used RNA interference applied non-invasively to knockdown Il1b and its endogenous antagonist Il1rn. We found that knocking down Il1b prior to pilocarpine injection increased the mortality rate of treated animals. Furthermore, we observed that, when exposing the animals to more Il1b by silencing its endogenous antagonist Il1rn, there was a better response to status epilepticus with decreased animal mortality in the acute phase of the PILO model. Thus, we show the feasibility of using a novel, less invasive approach to study genes involved in the inflammatory response in the central nervous system. Furthermore, our results provide suggestive evidence that modulating endogenous Il1b improves animal survival in the acute phase of the PILO model and may have effects that extend into the chronic phase.

Chemical and mechanical influence of root canal irrigation on biofilm removal from lateral morphological features of simulated root canals, dentine discs and dentinal tubules.

AIM: To investigate the anti-biofilm efficacy of irrigation using a simulated root canal model, the chemical effect of irrigants against biofilms grown on dentine discs and their impact on biofilm viscoelasticity, the efficacy of the irrigants in decontaminating infected dentinal tubules and the capacity of bacteria to regrow. METHODOLOGY: Biofilm removal, viscoelastic analysis of remaining biofilms and bacterial viability were evaluated using a simulated root canal model with lateral morphological features, dentine discs and a dentinal tubule model, respectively. Experiments were conducted using a two-phase irrigation protocol. Phase 1: a modified salt solution (RISA) and sodium hypochlorite (NaOCl) were used at a low flow rate to evaluate the chemical action of the irrigants. Ultrasonic activation (US) of a chemically inert solution (buffer) was used to evaluate the mechanical efficacy of irrigation. Phase 2: a final irrigation with buffer at a high flow rate was performed for all groups. Optical coherence tomography (OCT), low load compression testing (LLCT) and confocal scanning laser microscopy analysis were used in the different models. One-way analysis of variance (anova) was performed for the OCT and LLCT analysis, whilst Kruskal-Wallis and Wilcoxon ranked tests for the dentinal tubule model. RESULTS: US and high flow rate removed significantly more biofilm from the artificial lateral canal. For biofilm removal from the artificial isthmus, no significant differences were found between the groups. Within-group analysis revealed significant differences between the steps of the experiment, with the exception of NaOCl. For the dentine discs, no significant differences regarding biofilm removal and viscoelasticity were detected. In the dentinal tubule model, NaOCl exhibited the greatest anti-biofilm efficacy. CONCLUSIONS: The mechanical effect of irrigation is important for biofilm removal. An extra high flow irrigation rate resulted in greater biofilm removal than US in the artificial isthmus. The mechanical effect of US seemed to be more effective when the surface contact biofilm-irrigant was small. After the irrigation procedures, the remaining biofilm could survive after a 5-day period. RISA and NaOCl seemed to alter post-treatment remaining biofilms.

Biofilm removal from a simulated isthmus and lateral canal during syringe irrigation at various flow rates: a combined experimental and Computational Fluid Dynamics approach.

AIM: (i) To quantify biofilm removal from a simulated isthmus and a lateral canal in an artificial root canal system during syringe irrigation with NaOCl at different concentrations and delivered at various flow rates (ii) to examine whether biofilm removal is further improved by a final high-flow-rate rinse with an inert irrigant following irrigation with NaOCl. (iii) to simulate the irrigant flow in these areas using a computer model (iv) to examine whether the irrigant velocity calculated by the computer model is correlated to biofilm removal. METHODOLOGY: Ninety-six artificial root canals with either a simulated isthmus or lateral canal were used. A dual-species in vitro biofilm was formed in these areas using a Constant Depth Film Fermenter. NaOCl at various concentrations (2, 5 and 10%) or adhesion buffer (control) was delivered for 30 s by a syringe and an open-ended needle at 0.033, 0.083, or 0.166 mL s-1 or passively deposited in the main root canal (phase 1). All specimens were subsequently rinsed for 30 s with adhesion buffer at 0.166 mL s-1 (phase 2). The biofilm was scanned by Optical Coherence Tomography to determine the percentage of the remaining biofilm. Results were analysed by two 3-way mixed-design ANOVAs (α = 0.05). A Computational Fluid Dynamics model was used to simulate the irrigant flow inside the artificial root canal system. RESULTS: The flow rate during phase 1 and additional irrigation during phase 2 had a significant effect on the percentage of the remaining biofilm in the isthmus (P = 0.004 and P < 0.001). Additional irrigation during phase 2 also affected the remaining biofilm in the lateral canal significantly (P ≤ 0.007) but only when preceded by irrigation at medium or high flow rate during phase 1. The effect of NaOCl concentration was not significant (P > 0.05). Irrigant velocity in the isthmus and lateral canal increased with increasing flow rate and it was substantially correlated to biofilm removal from those areas. CONCLUSIONS: The irrigant flow rate affected biofilm removal in vitro more than NaOCl concentration. Irrigant velocity predicted by the computer model corresponded with the pattern of biofilm removal from the simulated isthmus and lateral canal.

The influence of time and irrigant refreshment on biofilm removal from lateral morphological features of simulated root canals.

AIM: To evaluate the effect of irrigant refreshment and exposure time of a 2% sodium hypochlorite solution (NaOCl) on biofilm removal from simulated lateral root canal spaces using two different flow rates. METHODOLOGY: A dual-species biofilm was formed by a Constant Depth Film Fermenter (CDFF) for 96 h in plug inserts with anatomical features resembling an isthmus or lateral canal-like structures. The inserts were placed in a root canal model facing the main canal. NaOCl 2% and demineralized water (control group) were used as irrigant solutions. Both substances were applied at a flow rate of 0.05 and 0.1 mL s-1 . The samples were divided into three groups with zero, one or two refreshments in a total exposure time of 15 min. A three-way analysis of variance (anova) was performed to investigate the interaction amongst the independent variables and the effect of consecutive irrigant refreshment on percentage of biofilm removal. A Tukey post hoc test was used to evaluate the effect of each independent variable on percentage biofilm removal in the absence of statistically significant interactions. RESULTS: For the lateral canal, NaOCl removed significantly more biofilm irrespective of the number of refreshments and exposure time (P = 0.005). There was no significant effect in biofilm removal between the consecutive irrigant refreshments measured in the same biofilm. For the isthmus, NaOCl removed significantly more biofilm irrespective of the number of refreshments and exposure time; both NaOCl and a high flow rate removed significantly more biofilm when the exposure time was analysed (P = 0.018 and P = 0.029, respectively). Evaluating the effect of consecutive irrigant refreshment on the same biofilm, 2% NaOCl, 0.1 mL s-1 flow rate and one or two refreshments removed significant more biofilm (P = 0.04, 0.034 and 0.003, <0.001, respectively). CONCLUSIONS: In this model, refreshment did not improve biofilm removal from simulated lateral root canal spaces. NaOCl removed more biofilm from the lateral canal- and isthmus-like structure. A higher flow rate removed significantly more biofilm from the isthmus-like structure. There was always remaining biofilm left after the irrigation procedures.

Antibacterial properties of silver nanoparticles as a root canal irrigant against Enterococcus faecalis biofilm and infected dentinal tubules.

AIM: To evaluate the antimicrobial action of an irrigant containing silver nanoparticles in an aqueous vehicle (AgNp), sodium hypochlorite and chlorhexidine against Enterococcus faecalis biofilm and infected dentinal tubules. METHODOLOGY: Bovine dentine blocks were used for E. faecalis biofilm development for 21 days and irrigated with 94 ppm AgNp solution, 2.5% NaOCl and 2% chlorhexidine for 5, 15 and 30 min. For infection of dentinal tubules with E. faecalis, dentine specimens from bovine incisors were submitted to a contamination protocol over 5 days, with eight centrifugation cycles on every alternate day, and irrigated with the same solutions and time intervals used for the biofilm. The specimens were stained with the Live/Dead technique and evaluated using a confocal laser scanning microscope (CLSM). The bioImage_L software was used for measurement of the total biovolume of biofilm in µm3 and percentage of viable bacteria (green cells) in biofilm and in dentinal tubules found after the irrigation. Statistical analyses were performed using Kruskal-Wallis and Dunn's tests for quantification of viable cells in biofilm, the Friedman test for comparisons of viable bacteria in dentinal tubules in different areas of the root canal and the Mann-Whitney U-test to compare the action of the irrigants between the two methods (P < 0.05). RESULTS: The AgNp solution eliminated fewer bacteria, but was able to dissolve more biofilm compared with chlorhexidine (P < 0.05). NaOCl had the greatest antimicrobial activity and biofilm dissolution capacity. AgNp solution had less antimicrobial action in infected dentinal tubules compared with NaOCl (P < 0.05). The AgNp solution after 5 min was more effective in eliminating planktonic bacteria in dentinal tubules than in biofilm, but at 30 min fewer viable bacteria were observed in the biofilm compared with intratubular dentine (P < 0.05). CONCLUSIONS: AgNp irrigant was not as effective against E. faecalis compared to solutions commonly used in root canal treatment. NaOCl is appropriate as an irrigant because it was effective in disrupting biofilm and in eliminating bacteria in biofilms and in dentinal tubules.

Influence of powder-to-water ratio on radiopacity, setting time, pH, calcium ion release and a micro-CT volumetric solubility of white mineral trioxide aggregate.

AIM: To evaluate the radiopacity, setting time, pH level, calcium ion release and solubility of white mineral trioxide aggregate (MTA; Angelus, Londrina, Pr, Brazil) with different powder-to-water ratios. METHODOLOGY: Three MTA groups were prepared using 4 : 1, 3 : 1 and 2 : 1 powder-to-water ratios. For the radiopacity analysis, the 10 × 1 mm specimens were arranged on occlusal films with a cylinder of dentine and an aluminium stepwedge. The digitized radiographs were evaluated with Digora 1.51 software to determine the radiographic density. The setting time test was performed according to the American Society for Testing and Materials 266/08 standard specifications, but the specimens were made according to International Organization for Standardization 6876:2001. Thirty acrylic teeth with root-end filling material were immersed in ultrapure water for measurement of pH level and calcium ion release (atomic absorption spectrophotometer) at 3, 24, 72 and 168 h. In the solubility test, the root-end fillings of 30 acrylic teeth were scanned twice by a Micro-CT, before and after immersion in ultrapure water for 168 h. Digital data were reconstructed, and the volume (mm(3) ) of the samples was obtained using CTan software (CTan v1.11.10.0, SkyScan). The data were statistically analysed by the anova, Tukey, Kruskal-Wallis and Dunn's tests. RESULTS: The radiopacity was higher (P < 0.05) when the 4 : 1 proportion was utilized. The setting time was longer, and the pH level and calcium ion release were higher (P < 0.05) with a greater volume of water. The group with more water (2 : 1) had significantly (P < 0.05) more material volume loss (6.46%) compared with the other groups. CONCLUSIONS: The ratio of powder/water significantly interfered with the physical and chemical properties of white MTA Angelus.

Nitrogen metabolism in lactating goats fed with diets containing different protein sources.

This study aimed to evaluate urea excretion, nitrogen balance and microbial protein synthesis in lactating goats fed with diets containing different protein sources in the concentrate (soybean meal, cottonseed meal, aerial part of cassava hay and leucaena hay). Four Alpine goats whose mean body weight was 42.6±6.1 kg at the beginning of the experiment, a mean lactation period of 94.0±9.0 days and a production of 1.7±0.4 kg of milk were distributed in a 4×4 Latin square with four periods of 15 days. Diets were formulated to be isonitrogenous, containing 103.0 g/kg of CP, 400 g/kg of Tifton 85 hay and 600 g/kg of concentrate. Diet containing cottonseed meal provided (p<0.05) increased excretion of urea and urea nitrogen in the urine (g/d and mg/kg of BW) when compared with leucaena hay. The diets affected the concentrations of urea nitrogen in plasma (p<0.05) and excretion of urea nitrogen in milk, being that soybean meal and cottonseed meal showed (p<0.05) higher than the average aerial part of the cassava hay. The use of diets with cottonseed meal as protein source in the concentrate in feeding of lactating goats provides greater nitrogen excretion in urine and negative nitrogen balance, while the concentrate with leucaena hay as a source of protein, provides greater ruminal microbial protein synthesis.

Functional and molecular characterization of kinin B1 and B 2 receptors in human bladder cancer: implication of the PI3Kγ pathway.

Kinins and their receptors have been recently implicated in cancer. Using functional and molecular approaches, we investigated the relevance of kinin B1 and B2 receptors in bladder cancer. Functional studies were conducted using bladder cancer cell lines, and human biopsies were employed for molecular studies. Both B1 des-Arg(9)-BK and B2 BK receptor agonists stimulated the proliferation of grade 3-derived T24 bladder cancer cells. Furthermore, treatment with B1 and B2 receptor antagonists (SSR240612 and HOE140) markedly inhibited the proliferation of T24 cells. Only higher concentrations of BK increased the proliferation of the grade 1 bladder cancer cell line RT4, while des-Arg(9)-BK completely failed to induce its proliferation. Real-time PCR revealed that the mRNA expression of kinin receptors, particularly B1 receptors, was increased in T24 cells relative to RT4 cells. Data from bladder cancer human biopsies revealed that B1 receptor expression was increased in all tumor samples and under conditions of chronic inflammation. We also show novel evidence demonstrating that the pharmacological inhibition of PI3Kγ (phosphatidylinositol 3-kinase) with AS252424, concentration-dependently reduced T24 cell proliferation induced by BK or des-Arg(9)-BK. Finally, the incubation of T24 cells with kinin agonists led to a marked activation of the PI3K/AKT and ERK 1/2 signaling pathways, whereas p38 MAP kinase remained unaffected. Kinin receptors, especially B1 receptors, appear to be implicated in bladder cancer progression. It is tempting to suggest that selective kinin antagonists might represent potential alternative therapies for bladder cancer.

A case-control study of protection against tuberculosis by BCG revaccination in Recife, Brazil.

SETTING: Metropolitan region of Recife, Brazil. OBJECTIVE: To estimate the additional protection against tuberculosis (TB) provided by a second dose of bacille Calmette-Guérin (BCG) vaccine. DESIGN: Case-control study. Cases were cases of TB newly diagnosed by the TB control programme, independent of clinical form. Three matched neighbourhood controls were selected using a systematic routine, starting from the case's address. The matching was within the age groups 7-9, 10-14 and 15-19 years. RESULTS: Analysis was conducted among 169 cases and 477 controls. For the efficacy of BCG revaccination against TB overall, matched (crude) vaccine effectiveness (VE) was -3 (95% CI -50-29) and matched (adjusted) VE was 8 (95% CI -77-52). CONCLUSIONS: This study suggests that a second dose of BCG does not offer additional protection. Revaccination should not be offered. As large numbers of subjects are already vaccinated and vaccine appears to offer some protection in older subjects, further studies with larger sample sizes could investigate the potential efficacy of revaccination with BCG in the age group > or = 15 years.

Lithium carbonate and coenzyme Q10 reduce cell death in a cell model of Machado-Joseph disease.

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by expansion of the polyglutamine domain of the ataxin-3 (ATX3) protein. MJD/SCA3 is the most frequent autosomal dominant ataxia in many countries. The mechanism underlying MJD/SCA3 is thought to be mainly related to protein misfolding and aggregation leading to neuronal dysfunction followed by cell death. Currently, there are no effective treatments for patients with MJD/SCA3. Here, we report on the potential use of lithium carbonate and coenzyme Q10 to reduce cell death caused by the expanded ATX3 in cell culture. Cell viability and apoptosis were evaluated by MTT assay and by flow cytometry after staining with annexin V-FITC/propidium iodide. Treatment with lithium carbonate and coenzyme Q10 led to a significant increase in viability of cells expressing expanded ATX3 (Q84). In addition, we found that the increase in cell viability resulted from a significant reduction in the proportion of apoptotic cells. Furthermore, there was a significant change in the expanded ATX3 monomer/aggregate ratio after lithium carbonate and coenzyme Q10 treatment, with an increase in the monomer fraction and decrease in aggregates. The safety and tolerance of both drugs are well established; thus, our results indicate that lithium carbonate and coenzyme Q10 are good candidates for further in vivo therapeutic trials.

Nitrogen balance of goat kids fed diets containing peach palm meal / [Balanço de nitrogênio em caprinos alimentados com dietas contendo farinha de pupunha]

O objetivo deste estudo foi avaliar o balanço de nitrogênio e a concentração de ureia no plasma em cabritos alimentados com dietas contendo farinha de pupunha em substituição ao milho (0, 10, 40, 60 e 85% MS). Trinta cabritos Boer × SRD, com 90 dias de idade e peso corporal inicial de 16,7 ± 3,5kg, foram distribuídos em um delineamento inteiramente ao acaso, com seis repetições. As dietas foram fornecidas diariamente ad libitum para permitir 10% das sobras. Três coletas foram realizadas a cada 28 dias. As dietas foram constituídas por milho, farelo de soja, farinha de pupunha, suplemento mineral e feno de Tifton-85, com a relação volumoso: concentrado de 30:70. O consumo de nitrogênio (N) diminuiu linearmente à medida que a farinha de pupunha substituiu o milho no concentrado. Com relação ao N digerido, os animais alimentados com níveis de farinha de pupunha apresentaram valores inferiores aos alimentados com o milho como única fonte de energia, com médias de 14,9 e 17,1g dia-1, respectivamente. A excreção urinária de N diminuiu à medida que a farinha de pupunha substituiu o milho no concentrado. O nível de 39% de farinha de pupunha reduziu a excreção urinária de ureia. Houve retenção máxima de N em 7,83g dia-1 para o nível de substituição de milho de 28,9% em farinha de pupunha.(AU)

Spinal blockage of P/Q- or N-type voltage-gated calcium channels modulates functional and symptomatic changes related to haemorrhagic cystitis in mice.

BACKGROUND AND PURPOSE: Spinal voltage-gated calcium channels (VGCCs) are pivotal regulators of painful and inflammatory alterations, representing attractive therapeutic targets. We examined the effects of epidural administration of the P/Q- and N-type VGCC blockers Tx3-3 and Phα1ß, respectively, isolated from the spider Phoneutria nigriventer, on symptomatic, inflammatory and functional changes allied to mouse cyclophosphamide (CPA)-induced haemorrhagic cystitis (HC). The effects of P. nigriventer-derived toxins were compared with those displayed by MVIIC and MVIIA, extracted from the cone snail Conus magus. EXPERIMENTAL APPROACH: HC was induced by a single i.p. injection of CPA (300 mg·kg(-1) ). Dose- and time-related effects of spinally administered P/Q and N-type VGCC blockers were assessed on nociceptive behaviour and macroscopic inflammation elicited by CPA. The effects of toxins were also evaluated on cell migration, cytokine production, oxidative stress, functional cystometry alterations and TRPV1, TRPA1 and NK1 receptor mRNA expression. KEY RESULTS: The spinal blockage of P/Q-type VGCC by Tx3-3 and MVIIC or N-type VGCC by Phα1ß attenuated nociceptive and inflammatory events associated with HC, including bladder oxidative stress and cytokine production. CPA produced a slight increase in bladder TRPV1 and TRPA1 mRNA expression, which was reversed by all the toxins tested. Noteworthy, Phα1ß strongly prevented bladder neutrophil migration, besides HC-related functional alterations, and its effects were potentiated by co-injecting the selective NK1 receptor antagonist CP-96345. CONCLUSIONS AND IMPLICATIONS: Our results shed new light on the role of spinal P/Q and N-type VGCC in bladder dysfunctions, pointing out Phα1ß as a promising alternative for treating complications associated with CPA-induced HC.

Rectal pulse granuloma.

Pulse granuloma is a rare benign entity, most likely representing a foreign body reaction to vegetable particles. We report a case of a pulse granuloma involving the rectum. The patient presented with a submucosal and intramuscular mass lesion found at routine rectal examination and subsequent colonoscopy. The mass was excised and the microscopic examination revealed acute and chronic inflammatory cells, foreign-body giant cells, vegetable matter, and convoluted hyaline rings and scattered circular structures containing basophilic granules, consistent with pulse granuloma. There are a few reports in the literature of pulse granulomas, with most occurring in the oral cavity or lungs. To the best of our knowledge, this is the first reported example of pulse granuloma in the rectum. Although rare, familiarity with this entity's distinctive histopathologic features may avoid a delay in diagnosis and prevent the expense of distinguishing it from its histologic lookalikes.

The spinal inhibition of N-type voltage-gated calcium channels selectively prevents scratching behavior in mice.

The present study investigated the effects of pharmacological spinal inhibition of voltage-gated calcium channels (VGCC) in mouse pruritus. The epidural administration of P/Q-type MVIIC or PhTx3.3, L-type verapamil, T-type NNC 55-0396 or R-type SNX-482 VGCC blockers failed to alter the scratching behavior caused by the proteinase-activated receptor 2 (PAR-2) activator trypsin, injected into the mouse nape skin. Otherwise, trypsin-elicited pruritus was markedly reduced by the spinal administration of preferential N-type VGCC inhibitors MVIIA and Phα1ß. Time-course experiments revealed that Conus magus-derived toxin MVIIA displayed significant effects when dosed from 1h to 4h before trypsin, while the anti-pruritic effects of Phα1ß from Phoneutria nigriventer remained significant for up to 12h. In addition to reducing trypsin-evoked itching, MVIIA or Phα1ß also prevented the itching elicited by intradermal (i.d.) injection of SLIGRL-NH2, compound 48/80 or chloroquine, although they did not affect H2O2-induced scratching behavior. Furthermore, the co-administration of MVIIA or Phα1ß markedly inhibited the pruritus caused by the spinal injection of gastrin-releasing peptide (GRP), but not morphine. Notably, the epidural administration of MVIIA or Phα1ß greatly prevented the chronic pruritus allied to dry skin model. However, either tested toxin failed to alter the edema formation or neutrophil influx caused by trypsin, whereas they significantly reduced the c-Fos activation in laminas I, II and III of the spinal cord. Our data bring novel evidence on itching transmission mechanisms, pointing out the therapeutic relevance of N-type VGCC inhibitors to control refractory pruritus.

Proline-induced changes in acetylcholinesterase activity and gene expression in zebrafish brain: reversal by antipsychotic drugs.

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures, cognitive dysfunctions, and schizoaffective disorders. However, the mechanisms related to these symptoms are still unclear. In the present study, we evaluated the in vivo and in vitro effects of proline on acetylcholinesterase (AChE) activity and gene expression in the zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0mM) during 1h or 7 days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000 µM) were tested. Long-term proline exposures significantly increased AChE activity for both treated groups when compared to the control (34% and 39%). Moreover, the proline-induced increase on AChE activity was completely reverted by acute administration of antipsychotic drugs (haloperidol and sulpiride), as well as the changes induced in ache expression. When assessed in vitro, proline did not promote significant changes in AChE activity. Altogether, these data indicate that the enzyme responsible for the control of acetylcholine levels might be altered after proline exposure in the adult zebrafish. These findings contribute for better understanding of the pathophysiology of hyperprolinemia and might reinforce the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.

Lithium carbonate and coenzyme Q10 reduce cell death in a cell model of Machado-Joseph disease

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by expansion of the polyglutamine domain of the ataxin-3 (ATX3) protein. MJD/SCA3 is the most frequent autosomal dominant ataxia in many countries. The mechanism underlying MJD/SCA3 is thought to be mainly related to protein misfolding and aggregation leading to neuronal dysfunction followed by cell death. Currently, there are no effective treatments for patients with MJD/SCA3. Here, we report on the potential use of lithium carbonate and coenzyme Q10 to reduce cell death caused by the expanded ATX3 in cell culture. Cell viability and apoptosis were evaluated by MTT assay and by flow cytometry after staining with annexin V-FITC/propidium iodide. Treatment with lithium carbonate and coenzyme Q10 led to a significant increase in viability of cells expressing expanded ATX3 (Q84). In addition, we found that the increase in cell viability resulted from a significant reduction in the proportion of apoptotic cells. Furthermore, there was a significant change in the expanded ATX3 monomer/aggregate ratio after lithium carbonate and coenzyme Q10 treatment, with an increase in the monomer fraction and decrease in aggregates. The safety and tolerance of both drugs are well established; thus, our results indicate that lithium carbonate and coenzyme Q10 are good candidates for further in vivo therapeutic trials.

Evaluation of DNA damage by the alkaline comet assay of the olfactory and respiratory epithelia of dogs from the city of São Paulo, Brazil.

Animals kept as pets may be considered sentinels for environmental factors to which humans could be exposed. Olfactory and respiratory epithelia are directly subjected to airborne factors, which could cause DNA lesions, and the alkaline comet assay is considered a reliable tool for the assessment of DNA damage. The objective of this work is to evaluate the extent of DNA damage by the comet assay of the olfactory and respiratory epithelia of dogs from different regions of the city of São Paulo, Brazil. Thirty-three clinically healthy dogs, aged 5 years or more, were used in the study, with 7 from the North region of São Paulo, 7 from the South region, 3 dogs from the East region, and 16 dogs from the West city region. Three dogs younger than 6 months were used as controls. DNA damage was analyzed by the alkaline comet assay. We observed no difference in histopathological analysis of olfactory and respiratory epithelia between dogs from different regions of São Paulo. Dogs older than 5 years presented significantly higher comet length in both olfactory and respiratory epithelia, when compared with controls, indicating DNA damage. When separated by regions, olfactory and respiratory epithelia presented similar DNA damage in dogs from different regions of São Paulo, corroborating with similar levels of particulate matter index (PM10) in all regions of the city. In this study, we report for the first time that the comet assay can be used to quantify the extent of DNA damage in dog olfactory and respiratory epithelia, and that comet length (DNA damage) increases with age, probably due to environmental factors. Air pollution, as measured by PM10, can be responsible for this DNA damage.

Cryptic anhydrobiotic potential in man: implications in medicine.

Anhydrobiosis ("life without water") is a state of suspended animation in a dry, ametabolic condition which some species enter before desiccation stress. This state confers resistance to other abiotic stresses such as radiation and heat. Once specimens are rehydrated life resumes. Application of such long-term storage in dry state without loss of viability would bring a considerable advance in organ transplant, maintenance of cell/tissue banks, vaccine storage and transport and many other medical applications. Since anhydrobiosis brings biological advantage to organisms, one would expect it to be positively selected during evolution. Moreover, our still limited understanding of the human genome, together with occasional discoveries of hidden endogenous processes, as well as reports of suspended animation in higher animals leads us to hypothesize the existence of cryptic anhydrobiosis in man. This hypothesis could be tested in laboratory animals and human-derived biological samples using several physical-chemical pre-conditioning/desiccation/rehydration parameters. Finding cryptic anhydrobiosis in human tissues would be a breakthrough in medicine providing means for long-term storage of biological samples in several stressful conditions.