Control of center of mass during gait of stroke patients: Statistical parametric mapping analysis.

BACKGROUND: The control of the center of mass is essential for a stable and efficient gait. Post-stroke patients present several impairments, which may compromise the control of the center of mass during gait in the sagittal and frontal planes. This study aimed to identify changes in the vertical and mediolateral behavior of the center of mass during the single stance phase of post-stroke patients using the statistical parametric mapping analysis. It also aimed to identify alterations in the center of mass trajectories regarding the motor recovery stages. METHODS: Seventeen stroke patients and 11 neurologically intact individuals were analyzed. The statistical parametric mapping approach was used to identify changes in the center of mass trajectories between stroke and healthy groups. The trajectories of the center of mass of post-stroke individuals were compared according to their motor recovery status. FINDINGS: A near-flat vertical trajectory of the center of mass was indenfitifed in the stroke group compared to their healthy counterparts, especially on the paretic side. The center of mass trajectories in both directions (vertical and mediolateral) presented substantial alteration at the end of the single stance phase in the stroke group. The trajectory of the center of mass of the stroke group was symmetrical in the mediolateral direction between the sides. The trajectories of the center of mass presented similar pattern irrespective of the motor recovery status. INTERPRETATION: The statistical parametric mapping approach showed to be suitable for determining gait changes in post-stroke individuals, irrespective of their motor recovery stage.

Strategies Adopted by Stroke Patients to Maintain Balance in Dynamic Tasks in a Video Game.

Postural instability affects motor tasks after a stroke. We investigated the strategies used to maintain balance during quiet standing posture and dynamic tasks in a video game. Sixteen stroke volunteers (12 males, 56 ± 9 years, post-stroke time 35 ± 10 months) and sixteen matched healthy volunteers had their biomechanical data collected to obtain the variables: center of mass, base of support, margin of stability, and weight symmetry. Healthy individuals and stroke patients showed similar dynamic stability. However, they adopted different motor strategies to achieve this: healthy individuals increased their base of support as they progressed to more challenging tasks, and stroke volunteers maintained the same base. The margin of stability of stroke volunteers was correlated with the MiniBEST scale.

Bovine genital leptospirosis: Evidence of ovarian infection by Leptospira interrogans.

Leptospirosis in ruminants causes reproductive failures leading to important economic losses. This study assessed the occurrence and genetically identified Leptospira spp. in the follicular fluid (FF) of naturally infected live cows. A total of 251 asymptomatic cows from different commercial dairy herds were subjected to ovum-pick up technique for follicular fluid sampling. PCR was performed for Leptospira spp. detection and phylogenetic analysis was later implemented for sequencing. From 251 samples analyzed, 67 (26.7 %) were lipL32-PCR positive, confirming the presence of leptospiral DNA on FF. Furthermore, it was possible to amplify and sequence nine strains after secY nested-PCR. All of them were identified as L. interrogans, with 100 % of identity with strains belonging to Sejroe serogroup. Our findings reveal a high occurrence of infection of Leptospira in the ovarium of asymptomatic cows, highlighting the importance of considering the silent leptospirosis syndrome when screening animals for assisted reproductive biotechniques.

Genome-wide promoter methylation profiling in a cellular model of melanoma progression reveals markers of malignancy and metastasis that predict melanoma survival.

The epigenetic changes associated with melanoma progression to advanced and metastatic stages are still poorly understood. To shed light on the CpG methylation dynamics during melanoma development, we analyzed the methylome profiles of a four-stage cell line model of melanoma progression: non-tumorigenic melanocytes (melan-a), premalignant melanocytes (4C), non-metastatic melanoma cells (4C11-), and metastatic melanoma cells (4C11+). We identified 540 hypo- and 37 hypermethylated gene promoters that together characterized a malignancy signature, and 646 hypo- and 520 hypermethylated promoters that distinguished a metastasis signature. Differentially methylated genes from these signatures were correlated with overall survival using TCGA-SKCM methylation data. Moreover, multivariate Cox analyses with LASSO regularization identified panels of 33 and 31 CpGs, respectively, from the malignancy and metastasis signatures that predicted poor survival. We found a concordant relationship between DNA methylation and transcriptional levels for genes from the malignancy (Pyroxd2 and Ptgfrn) and metastasis (Arnt2, Igfbp4 and Ptprf) signatures, which were both also correlated with melanoma prognosis. Altogether, this study reveals novel CpGs methylation markers associated with malignancy and metastasis that collectively could improve the survival prediction of melanoma patients.

Identification of vaginal Leptospira in cervical-vaginal mucus of slaughtered pigs in the amazon region.

Swine genital leptospirosis is an infectious disease that leads to economic losses due to abortions, stillbirths, and reproductive failures. Considering the scarcity of studies regarding this condition, the objective of the present study was to identify and analyse leptospires infecting the reproductive tract of female pigs slaughtered in the Amazon region. Cervical-vaginal mucus (CVM) from 150 non-pregnant females were collected and submitted to molecular analysis. Initially, polymerase chain reaction (PCR) based on the lipL32 gene was performed. A total of 26.7% (40/150) samples were positive, indicating the presence of Leptospira sp. DNA. Subsequently, positive lipL32-PCR samples were evaluated using secY nested-PCR and sequencing procedures. Eleven amplicons could be sequenced and were identified as Leptospira interrogans (100% identity). Results from phylogenetic analyses led to identification of a putative strain of L. interrogans serogroup Australis, which is indicative of this being a serogroup. In the present study, there was detection of female pigs with leptospires in CVM indicating the possibility of venereal transmission. The large number of genital positive cases could indicate that genital leptospirosis syndrome could also be relevant onto swine production.

The neurological fatigue index for stroke. Reliability of a Norwegian version.

OBJECTIVE: The aim of this study was to examine the test-retest reliability, internal consistency, and floor and ceiling effects of a Norwegian version of the Neurological Fatigue Index for Stroke (NFI-Stroke). METHOD: To evaluate the psychometric properties of the NFI-Stroke, persons with stroke were recruited. Inclusion criteria were: ≥18 years; a performance of ≥4 seconds on the Clock-Drawing Test; and participants had to speak and understand Norwegian. Test-retest reliability, internal consistency, and floor and ceiling effects were evaluated with Spearman's rho, Weighted Kappa, Cronbach's Alpha, corrected total-item correlation, percentage of the total score, and responses to each item. RESULT: Of 82 eligible, 66 respondents were included in the project. NFI-Stroke has a test-retest reliability of 0.89, 0.89, and 0.87 with Spearman's rho and 0.55-0.78 with Weighted Kappa. For the subgroup chronic stroke, Spearman's rho was 0.89, 0.86, and 0.93, and Weighted Kappa was 0.61-0.91. For the subgroup sub-acute stroke, Spearman's rho was 0.48, 0.55, and 0.51, and Weighted Kappa was 0.02-0.54. Cronbach's Alpha was 0.90. For the physical subscale, alpha was 0.89, and for the cognitive subscale 0.74. Corrected total-item correlation for NFI-Stroke was 0.50-0.78, 0.55-0.79 for the physical subscale, and 0.46-0.60 for the cognitive subscale. None scored the highest nor lowest possible score of the questionnaire. CONCLUSION: NFI-Stroke has high test-retest reliability, and high internal consistency with neither floor nor ceiling effects for persons with stroke. The questionnaire may be useful both in general rehabilitation in institutions as well as in the municipal health services.

Bovine Genital Leptospirosis and reproductive disorders of live subfertile cows under field conditions.

Bovine genital leptospirosis (BGL) is characterized by silent chronic reproductive disorders, most related to early embryonic death leading to estrus repetition, subfertility and abortions. However, most studies were conducted in slaughterhouses, which lacks reproductive and sanitary history of the studied animals. This study aimed to evaluate the occurrence of Leptospira sp. infection in live cows with history of low reproductive efficiency. Blood, urine, cervico-vaginal mucus and uterine fragment were collected from nine cows of the same herd presenting reproductive failure (abortions, estrus repetition and chronic infertility). Serology (MAT) and molecular analysis (PCR and nucleotide sequencing) were performed. Serology showed three (33.3%) seroreactive cows, two to Sejroe and one to Icterohaemorrhagiae serogroups. Six cows (66.7%) presented leptospiral DNA on genital samples, while all urine samples were negative. L. interrogans was identified in five samples, very closely related to strains from Sejroe (n = 3) and Icterohaemorrhagiae (n = 2) serogroups, while L. noguchii was identified in one sample. Results from this preliminary study demonstrates the presence of leptospires on uterus and reinforces the negative impact of leptospiral infection on reproductive tract, highlighting its association with reproductive failures on live animals.

Quantification of Global DNA Methylation in Canine Melanotic and Amelanotic Oral Mucosal Melanomas and Peripheral Blood Leukocytes From the Same Patients With OMM: First Study.

Oral mucosal melanomas (OMMs) are aggressive and resistant cancers of high importance in veterinary oncology. Amelanotic OMM produces comparatively less melanin and is considered to be more aggressive than melanotic OMM. Global DNA methylation profiles with hypomethylated or hypermethylated patterns have both been associated with aggressive neoplasms; however, global DNA hypomethylation seems to correlate to higher aggressiveness. Accordingly, global DNA methylation in peripheral blood leukocytes has been investigated to understand the role of systemic or environmental factors in cancer development. This study aimed to quantify global DNA methylation in canine melanotic and amelanotic OMM samples and in the peripheral blood leukocytes of the same dogs. Tumor tissue samples were collected from 38 dogs, of which 19 were melanotic and 19 were amelanotic OMM. These were submitted to immunohistochemistry (IHC) with anti-5-methylcytosine (5mC) and anti-Ki67 primary antibodies. Ki67- and 5mC-positive nuclei were manually scored with the help of an image analysis system. Peripheral blood samples were collected from 18 among the 38 OMM-bearing dogs and from 7 additional healthy control dogs. Peripheral blood leukocytes were isolated from the 25 dogs, and DNA was extracted and analyzed by high-performance liquid chromatography (HPLC) for global DNA methylation. The pattern of global DNA methylation in both canine melanotic and amelanotic OMM indicated higher percentages of weakly or negatively stained nuclei in most of the OMM cells, presuming predominant global DNA hypomethylation. In addition, Ki67 counts in amelanotic OMM were significantly higher than those in melanotic OMM (p < 0.001). Global DNA methylation different immunostaining patterns (strong, weak or negative) correlated with Ki67 scores. Global DNA methylation in circulating leukocytes did not differ between the 9 melanotic and 9 amelanotic OMM or between the 18 OMM-bearing dogs and the 7 healthy dogs. This study provides new information on canine melanotic and amelanotic OMM based on global DNA methylation and cell proliferation.

Physiotherapy access for children and adolescents with physical disabilities in public institutions. / Acesso à fisioterapia de crianças e adolescentes com deficiência física em instituições públicas.

This study aims to identify the factors that hinder physically disabled children and adolescent's access to physiotherapy in establishments accredited by the Unified Health System in Curitiba, Paraná, Brazil. This is a cross-sectional study that interviewed managers, physiotherapists, parents or guardians of children with physical disabilities. The perception of difficulties of each interviewed group was compared through descriptive statistics, considering the following realms: availability, purchasing power, information and acceptability. Considering the realm of availability, we identified the following hindrances: distance between the physiotherapy location and user's residence, inadequate supply of services and waiting list. Regarding the purchasing power, we observed the lack of financial resources among parents. Regarding information and acceptability, the lack of information about diagnosis and denial were reported by participants. We can conclude that the physiotherapy service has not been timely assured to children's motor development, so it is necessary to review the organization and distribution of services, and effective policies are required to ensure their use.

Este estudo objetiva identificar os fatores que dificultam o acesso de crianças e adolescentes com deficiência física ao tratamento fisioterapêutico, em estabelecimentos credenciados ao Sistema Único de Saúde em Curitiba, Paraná, Brasil. Trata-se de um estudo transversal que entrevistou gestores, fisioterapeutas, pais e/ou responsáveis por crianças com deficiência física. A percepção de dificuldades de cada um dos grupos de entrevistados foi comparada por meio de estatísticas descritivas, considerando as dimensões: disponibilidade, poder de pagamento, informação e aceitabilidade. Considerando a dimensão disponibilidade, identificaram-se como dificuldades: distância entre o estabelecimento e a residência dos usuários, pouca oferta de serviços e fila de espera. Em relação ao poder de pagamento, observou-se a falta de recursos financeiros entre os pais. Nas dimensões informação e aceitabilidade, a falta de informação acerca do diagnóstico e a negação foram referidas pelos participantes. Conclui-se que o serviço de fisioterapia não tem sido garantido em tempo favorável ao desenvolvimento motor das crianças, de modo que se faz necessário rever a organização e distribuição dos serviços, bem como são essenciais políticas efetivas que garantam a sua utilização.

Acesso à fisioterapia de crianças e adolescentes com deficiência física em instituições públicas / Physiotherapy access for children and adolescents with physical disabilities in public institutions

Resumo Este estudo objetiva identificar os fatores que dificultam o acesso de crianças e adolescentes com deficiência física ao tratamento fisioterapêutico, em estabelecimentos credenciados ao Sistema Único de Saúde em Curitiba, Paraná, Brasil. Trata-se de um estudo transversal que entrevistou gestores, fisioterapeutas, pais e/ou responsáveis por crianças com deficiência física. A percepção de dificuldades de cada um dos grupos de entrevistados foi comparada por meio de estatísticas descritivas, considerando as dimensões: disponibilidade, poder de pagamento, informação e aceitabilidade. Considerando a dimensão disponibilidade, identificaram-se como dificuldades: distância entre o estabelecimento e a residência dos usuários, pouca oferta de serviços e fila de espera. Em relação ao poder de pagamento, observou-se a falta de recursos financeiros entre os pais. Nas dimensões informação e aceitabilidade, a falta de informação acerca do diagnóstico e a negação foram referidas pelos participantes. Conclui-se que o serviço de fisioterapia não tem sido garantido em tempo favorável ao desenvolvimento motor das crianças, de modo que se faz necessário rever a organização e distribuição dos serviços, bem como são essenciais políticas efetivas que garantam a sua utilização.

Abstract This study aims to identify the factors that hinder physically disabled children and adolescent's access to physiotherapy in establishments accredited by the Unified Health System in Curitiba, Paraná, Brazil. This is a cross-sectional study that interviewed managers, physiotherapists, parents or guardians of children with physical disabilities. The perception of difficulties of each interviewed group was compared through descriptive statistics, considering the following realms: availability, purchasing power, information and acceptability. Considering the realm of availability, we identified the following hindrances: distance between the physiotherapy location and user's residence, inadequate supply of services and waiting list. Regarding the purchasing power, we observed the lack of financial resources among parents. Regarding information and acceptability, the lack of information about diagnosis and denial were reported by participants. We can conclude that the physiotherapy service has not been timely assured to children's motor development, so it is necessary to review the organization and distribution of services, and effective policies are required to ensure their use.

The effects of stretching on muscle morphometry of ovariectomized rats / Efeitos do alongamento na morfometria do músculo em ratas ovarectomizadas / Los efectos del ejercicio de estiramiento en la morfometria muscular de las ratas ovariectomizadas

Abstract Introduction: Ageing is responsible for structural alterations, declining of all physiological variables, including range of motion and skeletal muscle function, known as sarcopenia. Objective: The aim of the study was to evaluate the effects of stretching on muscle morphometry in ovariectomized rats. Method: 21 female Wistar rats (12 weeks, 218 ± 22 g) were divided into 4 groups: control (CONTROL, n = 3) intact; ovariectomized and hysterectomized (OH, n = 6); Stretching (STRET, n = 6); ovariectomized and hysterectomized and stretching (OHS, n = 6). The rats were subjected to ovariectomy and hysterectomy. The stretching protocol of the soleus muscle lasted 10 repetitions of 1 minute with 45s interval between each repetition performed 3 times a week for 3 weeks. After 3 weeks, the rats were weighed and the muscles of both hind limbs were removed weighed and analyzed at muscle length; serial sarcomere number; sarcomere length; muscle fiber cross-sectional area (MFCSA) and percentage of connective tissue. Results: The final body weight increased in all groups. The serial sarcomere number of STRET was greater than the OH. The muscle fibers' cross-sectional area of OHS was higher than CONTROL. Conclusion: It can be concluded that ovariectomy and hysterectomy prevented sarcomerogenesis even when stretching was applied. However, the stretching protocol enhanced muscle trophismof ovariectomized and hysterectomized rats. It might be suggested that longitudinal growth (serial sarcomeres) and radial (ASTFM) are differently regulated by stretching in intact and/or estrogen depleted (ovariectomy and hysterectomy) skeletal muscle.

Resumo Introdução: O envelhecimento é responsável por alterações estruturais, com declínio de todas as variáveis fisiológicas, incluindo amplitude de movimento e função muscular esquelética. Objetivo: O objetivo do estudo foi avaliar os efeitos do alongamento na morfometria muscular de ratas ovariectomizadas. Método: Assim, 21 ratas Wistar (12 semanas, 218 ± 22 g) foram divididas em 4 grupos: Controle (CONTROL, n = 3) intacto; Ovariectomizadas e histerectomizadas (OH, n = 6); Alongamento (ALONG, n = 6); Ovariectomizadas e histerectomizadas e Alongamento (OHA, n = 6). As ratas foram submetidas a ovariectomia e histerectomia. O alongamento do músculo sóleo foi composto de 10 repetições de 1 minuto com intervalo de 45s entre cada repetição, realizado 3 vezes por semana, durante 3 semanas. Após 3 semanas, as ratas foram pesadas e os músculos sóleos de ambas as patas foram retirados, pesados e analisados: comprimento do músculo; número de sarcômeros em série; comprimento do sarcômero; área da seção transversal das fibras musculares (ASTFM) e porcentagem de tecido conjuntivo. Resultados: O peso corporal final aumentou em todos os grupos. O número de sarcômeros em série do ALONG foi maior que o OH. A área de secção transversa das fibras musculares do OHA foi superior ao CONTROL. Conclusão: Conclui-se que a ovariectomia e histerectomia impediram a sarcomerogênese mesmo quando realizado alongamento. Porém, o protocolo de alongamento incrementou o trofismo muscular em ratas ovarietomizadas e histerectomizadas. Sugere-se que o crescimento longitudinal (número sarcômeros em série) e radial (ASTFM) respondem diferentemente ao alongamento em músculo esquelético intacto e/ou com depleção estrogênio (ovarietomia e histerectomia).

Resumen Introducción: El envejecimiento es responsable de cambios estructurales del organismo, con disminución de todas las variables fisiológicas, incluyendo la amplitud de movimiento y función muscular esquelética, conocida como sarcopenia. Objetivo: El objetivo del estudio fue evaluar los efectos de los ejercicios de estiramiento en la morfometria muscular de ratas ovarectomizadas. Método: Fueron seleccionadas 21 ratas Wistar (12 semanas, 218 ± 22g) que fueron divididas en 4 grupos: control (CONTROL, n = 3) intacto; ovarectomizadas y histerectomizadas (OH, n = 6); estiramiento (ALONG, n = 6); ovarectomizadas y histerectomizadas y estiramiento (OHA, n = 6). Las ratas han sido sometidas a ovariectomía y histerectomía. El protocolo de estiramiento del músculo sóleo fue realizado en 10 repeticiones de 1 minuto con intervalos de 45 segundos entre cada repetición, realizada 3 veces por semana, durante 3 semanas. Después de 3 semanas, las ratas fueron pesadas y los músculos de ambas patas fueron retirados para analizar el peso muscular; la longitud del músculo; número de sarcómera en serie; longitud de sarcómera; área de la sección transversal de las fibras musculares (ASTFM) y porcentaje del tejido conjuntivo. Resultado: El peso corporal final aumentó en todos los grupos. El número de sarcómera en serie de ALONG fue mayor que el OH. El área de la sección transversal de las fibras musculares del OHA fue superior al de CONTROL. Conclusión: En conclusión, ovariectomía y histerectomía impiedieron la sarcomerogénesis mismo cuando se realizó el estiramiento. Sin embago, el estiramiento aumentó el trofismo muscular de las ratas ovarectomizadas y histerectomizadas. Sugiere que el crescimiento longitudinal (número sarcômeros) y radial (área de sección transversal) responden diferentemente al estiramiento en músculo esqueletico intacto y/o con depleción estrógeno (ovariectomía y histerectomía).

Anhydroecgonine methyl ester (AEME), a cocaine pyrolysis product, impairs glutathione-related enzymes response and increases lipid peroxidation in the hippocampal cell culture.

Crack cocaine smokers inhale, alongside with cocaine, its pyrolysis product, anhydroecgonine methyl ester (AEME). We have previously described AEME neurotoxic effect and its additive effect when co-incubated with cocaine. Our aim was to evaluate, the effect of AEME, cocaine and AEME-cocaine combination on glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activities after 3 and 6 h of exposure, periods previous to neuronal death. Lipid peroxidation was evaluated through malonaldehyde (MDA) levels at 3, 6, 24 and 48 h of exposure. All treated groups reduced neuronal viability after 24 h of exposure. AEME and cocaine decreased GPx, GR and GST activities after 3 and 6 h, with an increase in MDA levels after 48 h. AEME-cocaine combination decreased the enzymes activities after 3 and 6 h, showing an additive effect in MDA levels after 48 h. These data show that the glutathione-related enzymes imbalance caused by AEME, cocaine or AEME-cocaine combination exposure preceded neuronal death and lipid peroxidation. Moreover, the additive effect on lipid peroxidation observed with AEME-cocaine exposure after 48 h, suggest a higher neurotoxic effect after crack cocaine use when compared to cocaine alone.

Influência da Dança Sênior no Equilíbrio de Indivíduos com Doença de Parkinson / Influence of Senior Dancing on the Balance of Individuals with Parkinson's Disease / Influencia de la danza para adultos mayores en el equilibrio de las personas con enfermedad de Parkinson

A Doença de Parkinson (DP) pode apresentar déficits ao idoso quanto aos ajustes motores para manter o equilíbrio funcional. O estudo avaliou o equilíbrio de uma amostra de 34 indivíduos idosos com DP divididos em dois grupos, fisioterapia convencional (GF = 17) e fisioterapia e dança sênior (GD =17). Como instrumento de avaliação, foram utilizadas: a Escala Unificada de Avaliação para Doença de Parkinson, a Escala de Equilíbrio de Berg e o índice de Tinetti. Para a amostra avaliada, não houve diferença significativa entre os resultados dos grupos GD e GF em relação ao equilíbrio dinâmico nos escores dos instrumentos de avaliação aplicados.

Parkinson's disease (PD) may present motor deficits to the elderly to maintain functional balance. The study evaluated the balance of the elderly with PD through a sample of 34 individuals divided into 2 groups, conventional physiotherapy (GF = 17) and physiotherapy and senior dance (GD = 17). The UPDRS, EEB and Tinetti scales were used in GF and GD. For the evaluation sample, there was no difference between DS and the GF about the balance in the scores of the applied scales.

La enfermedad de Parkinson (EP) puede presentar déficits en los ancianos en términos de ajustes motores para mantener el equilibrio funcional. El estudio evaluó el equilibrio de las personas mayores con EP a través de una muestra de 34 individuos divididos en 2 grupos, fisioterapia convencional (FG = 17) y fisioterapia y danza para adultos mayores (DG = 17). Las escalas UPDRS, EEB y Tinetti se utilizaron en GF y GD. Para la muestra evaluada, no hubo diferencia entre DS y el FG en relación con el equilibrio en las puntuaciones de las escalas aplicadas.

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter.

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2'-deoxyguanosine (1,N2-εdGuo) and 1,N6-etheno-2'-deoxyadenosine (1,N6-εdAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present. We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

Global DNA methylation of peripheral blood leukocytes from dogs bearing multicentric non-Hodgkin lymphomas and healthy dogs: A comparative study.

Non-Hodgkin lymphomas are among the most common types of tumors in dogs, and they are currently accepted as comparative models of the disease in humans. Aberrant patterns of DNA methylation seem to play a key role in the development of hematopoietic neoplasms in humans, constitute a special mechanism of transcriptional control, and may be influenced by genetic and environmental factors. Blood leukocyte DNA global methylation has been poorly investigated in dogs. The aim of this study is to examine whether peripheral blood global DNA methylation is associated with canine multicentric lymphomas. Peripheral venous blood samples from ten healthy dogs and nine dogs bearing multicentric lymphomas were collected, and the buffy coat was separated. Global DNA methylation was analyzed by High Performance Liquid Chromatography (HPLC) and immunocytochemistry (ICC). In both analyses, leukocytes from dogs with lymphoma presented lower global DNA methylation than in healthy dogs (HPLC: p = 0.027/ 5MeCyt immunoreactivity scores: p = 0.015). Moderate correlation was observed between the results obtained by HPLC and ICC (correlation coefficient = 0.50). For the identification of differently methylated genes between both groups, the Infinium Human Methylation (HM) EPIC BeadChip (850K) was used. Of the 853,307 CpGs investigated in the microarray, there were 34,574 probes hybridized in the canine samples. From this total, significant difference was observed in the methylation level of 8433 regions, and through the homologous and orthologous similarities 525 differently methylated genes were identified between the two groups. This study is pioneer in suggesting that dogs bearing non-Hodgkin lymphoma presented DNA global hypomethylation of circulating leukocytes compared with healthy dogs. Although canine samples were used in an assay developed specifically for human DNA, it was possible to identify differently methylated genes and our results reiterate the importance of the use of peripheral blood leukocytes in cancer research and possible new biomarkers targets.

Anhydroecgonine methyl ester (AEME), a cocaine pyrolysis product, impairs glutathione-related enzymes response and increases lipid peroxidation in the hippocampal cell culture

Crack cocaine smokers inhale, alongside with cocaine, its pyrolysis product, anhydroecgonine methyl ester (AEME). We have previously described AEME neurotoxic effect and its additive effect when co-incubated with cocaine. Our aim was to evaluate, the effect of AEME, cocaine and AEME-cocaine combination on glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activities after 3 and 6h of exposure, periods previous to neuronal death. Lipid peroxidation was evaluated through malonaldehyde (MDA) levels at 3, 6, 24 and 48h of exposure. All treated groups reduced neuronal viability after 24h of exposure. AEME and cocaine decreased GPx, GR and GST activities after 3 and 6h, with an increase in MDA levels after 48h. AEME-cocaine combination decreased the enzymes activities after 3 and 6h, showing an additive effect in MDA levels after 48h. These data show that the glutathione-related enzymes imbalance caused by AEME, cocaine or AEME-cocaine combination exposure preceded neuronal death and lipid peroxidation. Moreover, the additive effect on lipid peroxidation observed with AEME-cocaine exposure after 48h, suggest a higher neurotoxic effect after crack cocaine use when compared to cocaine alone.

Anhydroecgonine methyl ester (AEME), a cocaine pyrolysis product, impairs glutathione-related enzymes response and increases lipid peroxidation in the hippocampal cell culture

Crack cocaine smokers inhale, alongside with cocaine, its pyrolysis product, anhydroecgonine methyl ester (AEME). We have previously described AEME neurotoxic effect and its additive effect when co-incubated with cocaine. Our aim was to evaluate, the effect of AEME, cocaine and AEME-cocaine combination on glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activities after 3 and 6h of exposure, periods previous to neuronal death. Lipid peroxidation was evaluated through malonaldehyde (MDA) levels at 3, 6, 24 and 48h of exposure. All treated groups reduced neuronal viability after 24h of exposure. AEME and cocaine decreased GPx, GR and GST activities after 3 and 6h, with an increase in MDA levels after 48h. AEME-cocaine combination decreased the enzymes activities after 3 and 6h, showing an additive effect in MDA levels after 48h. These data show that the glutathione-related enzymes imbalance caused by AEME, cocaine or AEME-cocaine combination exposure preceded neuronal death and lipid peroxidation. Moreover, the additive effect on lipid peroxidation observed with AEME-cocaine exposure after 48h, suggest a higher neurotoxic effect after crack cocaine use when compared to cocaine alone.

Genotoxic and epigenotoxic effects in mice exposed to concentrated ambient fine particulate matter (PM2.5) from São Paulo city, Brazil.

BACKGROUND: The Metropolitan Area of São Paulo has a unique composition of atmospheric pollutants, and positive correlations between exposure and the risk of diseases and mortality have been observed. Here we assessed the effects of ambient fine particulate matter (PM2.5) on genotoxic and global DNA methylation and hydroxymethylation changes, as well as the activities of antioxidant enzymes, in tissues of AJ mice exposed whole body to ambient air enriched in PM2.5, which was concentrated in a chamber near an avenue of intense traffic in São Paulo City, Brazil. RESULTS: Mice exposed to concentrated ambient PM2.5 (1 h daily, 3 months) were compared to in situ ambient air exposed mice as the study control. The concentrated PM2.5 exposed group presented increased levels of the oxidized nucleoside 8-oxo-7,8-dihydro-2'-deoxyguanosine in lung and kidney DNA and increased levels of the etheno adducts 1,N6-etheno-2'-deoxyadenosine and 1,N2-etheno-2'-deoxyguanosine in kidney and liver DNA, respectively. Apart from the genotoxic effects, the exposure to PM2.5 led to decreased levels of the epigenetic mark 5-hydroxymethylcytosine (5-hmC) in lung and liver DNA. Changes in lung, liver, and erythrocyte antioxidant enzyme activities were also observed. Decreased glutathione reductase and increased superoxide dismutase (SOD) activities were observed in the lungs, while the liver presented increased glutathione S-transferase and decreased SOD activities. An increase in SOD activity was also observed in erythrocytes. These changes are consistent with the induction of local and systemic oxidative stress. CONCLUSIONS: Mice exposed daily to PM2.5 at a concentration that mimics 24-h exposure to the mean concentration found in ambient air presented, after 3 months, increased levels of DNA lesions related to the occurrence of oxidative stress in the lungs, liver, and kidney, in parallel to decreased global levels of 5-hmC in lung and liver DNA. Genetic and epigenetic alterations induced by pollutants may affect the genes committed to cell cycle control, apoptosis, and cell differentiation, increasing the chance of cancer development, which merits further investigation.

Oxidation of 1-N2-etheno-2'-deoxyguanosine by singlet molecular oxygen results in 2'-deoxyguanosine: a pathway to remove exocyclic DNA damage?

Exocyclic DNA adducts are considered as potential tools for the study of oxidative stress-related diseases, but an important aspect is their chemical reactivity towards oxidant species. We report here the oxidation of 1-N2-etheno-2'-deoxyguanosine (1,N2-εdGuo) by singlet molecular oxygen (1O2) generated by a non-ionic water-soluble endoperoxide [N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide endoperoxide (DHPNO2)] and its corresponding oxygen isotopically labeled [18O]-[N,N'-di(2,3-dihydroxypropyl)-1,4- naphthalenedipropanamide endoperoxide (DHPN18O2)], and by photosensitization with two different photosensitizers [methylene blue (MB) and Rose Bengal (RB)]. Products detection and characterization were achieved using high performance liquid chromatography (HPLC) coupled to ultraviolet and electrospray ionization (ESI) tandem mass spectrometry, and nuclear magnetic resonance (NMR) analyses. We found that dGuo is regenerated via reaction of 1O2 with the ε-linkage, and we propose a dioxetane as an intermediate, which cleaves and loses the aldehyde groups as formate residues, or alternatively, it generates a 1,2-ethanediol adduct. We also report herein the quenching rate constants of 1O2 by 1,N2-εdGuo and other etheno modified nucleosides. The rate constant (kt) values obtained for etheno nucleosides are comparable to the kt of dGuo. From these results, we suggest a possible role of 1O2 in the cleanup of etheno adducts by regenerating the normal base.

Detection of bovine carriers of Leptospira by serological, bacteriological, and molecular tools.

Bovine leptospirosis is an important infectious disease that causes reproductive problems and economic risks, particularly in the tropics. The present study aimed to determine the extent of Leptospira infection among bovines on a slaughterhouse from Rio de Janeiro, Brazil via serological, bacteriological, and molecular tests. Two hundred eight bovines were examined in total, and we obtained 208 blood samples for serology, 198 urine samples collected via direct bladder puncture for polymerase chain reaction (PCR) and culture, 208 kidney samples (one from each animal) for PCR and culture, and 92 vaginal fluid samples from sterile swabs for PCR and culture. Serology demonstrated that 77/208 (37%) of the animals presented anti-Leptospira antibodies. Serogroup Sejroe was by far the most common. One hundrd thirty-three animals (63.9%) were PCR positive in at least one of the tested samples and were considered as Leptospira carriers. Furthermore, ten isolates were obtained by pure culture, all of them from urine samples. Bovine leptospirosis is widely prevalent, and the occurrence of renal carriers was unexpectedly much higher than generally reported.